In vivo production and delivery of erthropoietin or insulinotropin for gene therapy

ABSTRACT

The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes erythropoietin or an insulinotropin [e.g., derivatives of glucagon-like peptide 1 (GLP-1)], methods by which primary and secondary cells are transfected to include exogenous genetic material encoding erythropoietin or an insulinotropin, methods of producing clonal cell strains or heterogenous cell strains which express eruthropoietin or an insulinotropin, methods of gene therapy in which the transfected primary or secondary cells are used, and methods of producing antibodies using the transfected primary or secondary cells.  
     The present invention includes primary and secondary somatic cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, glial cells, neural cells, formed elements of the blood, muscle cells, other somatic cells which can be cultured and somatic cell precursors, which have been transfected with exogenous DNA encoding EPO or an insulinotropin, which is stably integrated into their genomes or is expressed in the cells episomally.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.07/787,840, filed Nov. 5, 1991, entitled “In Vivo Protein Production andDelivery System for Gene Therapy” and of U.S. Ser. No. 07/789,188, filedNov. 5, 1991, entitled “Targeted Introduction of DNA Into Primary orSecondary Cells and Their Use for Gene Therapy”. The teachings of theseapplications are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] A variety of congenital, acquired, or induced syndromes areassociated with insufficient numbers of erythrocytes (red blood cells orRBCs). The clinical consequence of such syndromes, collectively known asthe anemias, is a decreased oxygen-carrying potential of the blood,resulting in fatigue, weakness, and failure-to-thrive. Erythropoietin(EPO), a glycoprotein of molecular mass 34,000 daltons, is synthesizedand released into the systemic circulation in response to reduced oxygentension in the blood. EPO, primarily synthesized in the kidney and, to alesser extent, in the liver, acts on erythroid precursor cells [ColonyForming Units-Erythroid (CFU-E) and Burst-Forming Units-Erythroid(BFU-E)] to promote differentiation into reticulocytes and, ultimately,mature erythrocytes.

[0003] The kidney is the major site of EPO production and, thus, renalfailure or nephrectomy can lead to decreased EPO synthesis, reduced RBCnumbers, and, ultimately, severe anemia as observed in predialysis anddialysis patients. Subnormal-RBC counts may also result from the toxiceffects of chemotherapeutic agents or azidothymidine (AZT) (used in thetreatment of cancers and AIDS, respectively) on erythroid precursorcells. In addition, a variety of acquired and congenital syndromes, suchas aplastic anemia, myeloproliferative syndrome, malignant lymphomas,multiple myeloma, neonatal prematurity, sickle-cell anemia, porphyriacutanea tarda, and Gaucher's disease include anemia as one clinicalmanifestation of the syndrome.

[0004] Purified human EPO or recombinant human EPO may be administeredto patients in order to alleviate anemia by increasing erythrocyteproduction. Typically, the protein is administered by regularintravenous injections. The administration of EPO by injection is animperfect treatment. Normal individuals maintain a relatively constantlevel of EPO, which is in the range of 6-30 mU/ml, depending on theassay used. After typical treatment regimens, serum EPO levels may reach3,000-5,000 mU/Ml following a single injection, with levels falling overtime as the protein is cleared from the blood.

[0005] If a relatively constant level of EPO is to be provided in theblood (i.e., to mimic the normal physiology of the protein), a deliverysystem that is capable of releasing a continuous, precisely dosedquantity of EPO into the blood is necessary.

SUMMARY OF THE INVENTION

[0006] The present invention relates to transfected primary andsecondary somatic cells of vertebrate origin, particularly mammalianorigin, transfected with exogenous genetic material (DNA or RNA) whichencodes a clinically useful product, such as erythropoietin (EPO) orinsulinotropin [e.g. derivatives of glucagon-like peptide 1 (GLP-1) suchas GLP(7-37), GLP(7-36), GLP-1(7-35) and GLP-1(7-34) as well as theircarboxy-terminal amidated derivatives produced by in vivo amidatingenzymes and derivatives which have amino acid alterations or otheralterations which result in substantially the same biological activityor stability in the blood as that of a truncated GLP-1 or enhancedbiological activity or stability], methods by which primary andsecondary cells are transfected to include exogenous genetic materialencoding EPO or insulinotropin, methods of producing clonal cell strainsor heterogenous cell strains which express exogenous genetic materialencoding EPO or insulinotropin, a method of providing EPO orinsulinotropin in physiologically useful quantitites to an individual inneed thereof, through the use of transfected cells of the presentinvention or by direct injection of DNA encoding EPO into an individual;and methods of producing antibodies against the encoded product usingthe transfected primary or secondary cells. Transfected cells containingEPO-encoding exogenous genetic material express EPO and, thus, areuseful for preventing or treating conditions in which EPO productionand/or utilization are inadequate or compromised, such as in anycondition or disease in which there is anemia. Similarly, transfectedcells containing insulinotropin-encoding exogenous genetic materialexpress insulino-tropin and, thus, are useful for treating individualsin whom insulin secretion, sensitivity or function is compromised (e.g.,individuals with insulin-dependent or non-insulin dependent diabetes).

[0007] The present invention includes primary and secondary somaticcells, such as fibroblasts, keratinocytes, epithelial cells, endothelialcells, glial cells, neural cells, formed elements of the blood, musclecells, other somatic cells which can be cultured and somatic cellprecursors, which have been transfected with exogenous DNA encoding EPOor exogenous DNA encoding insulinotropin. The exogenous DNA is stablyintegrated into the cell genome or is expressed in the cells episomally.The exogenous DNA encoding EPO is introduced into cells operativelylinked with additional DNA sequences sufficient for expression of EPO intransfected cells. The exogenous DNA encoding EPO is preferably DNAencoding human EPO but, in some instances, can be DNA encoding mammalianEPO of non-human origin. EPO produced by the cells is secreted from thecells and, thus, made available for preventing or treating a conditionor disease (e.g., anemia) in which EPO production and/or utilization isless than normal or inadequate for maintaining a suitable level of RBCs.Cells produced by the present method can be introduced into an animal,such as a human, in need of EPO and EPO produced in the cells issecreted into the systemic circulation. As a result, EPO is madeavailable for prevention or treatment of a condition in which EPOproduction and/or utilization is less than normal or inadequate tomaintain a suitable level of RBCs in the individual. Similarly,exogenous DNA encoding insulinotropin is introduced into cellsoperatively linked with additional DNA sequences sufficient forexpression of insulinotropin in transfected cells. The encodedinsulinotropin is made available to prevent or treat a condition inwhich insulin production or function is compromised or glucagon releasefrom the pancreas is to be inhibited.

[0008] Primary and secondary cells transfected by the subject method canbe seen to fall into three types or categories: 1) cells which do not,as obtained, produce and/or secrete the encoded protein (e.g., EPO,insulinotropin; 2) cells which produce and/or secrete the encodedprotein (e.g., EPO, insulinotropin) but in lower quantities than normal(in quantities less than the physiologically normal lower level) or indefective form, and 3) cells which make the encoded protein (e.g., EPOor insulinotropin) at physiologically normal levels, but are to beaugmented or enhanced in their production and/or secretion of theencoded protein.

[0009] Exogenous DNA encoding EPO is introduced into primary orsecondary cells by a variety of techniques. For example, a constructwhich includes exogenous DNA encoding EPO and additional DNA sequencesnecessary for expression of EPO in recipient cells is introduced intoprimary or secondary cells by electroporation, microinjection, or othermeans (e.g., calcium phosphate precipitation, modified calcium phosphateprecipitation, polybrene precipitation, microprojectile bombardment,liposome fusion, receptor-mediated DNA delivery). Alternatively, avector, such as a retroviral vector, which includes exogenous DNAencoding EPO can be used, and cells can be genetically modified as aresult of infection with the vector. Similarly, exogenous DNA encodinginsulinotropin is introduced into primary or secondary cells using oneof a variety of methods.

[0010] In addition to exogenous DNA encoding EPO or insulinotropin,transfected primary and secondary cells may optionally contain DNAencoding a selectable marker, which is expressed and confers uponrecipient cells a selectable phenotype, such as antibiotic resistance,resistance to a cytotoxic agent, nutritional prototrophy or expressionof a surface protein. Its presence makes it possible to identify andselect cells containing the exogenous DNA. A variety of selectablemarker genes can be used, such as neo, gpt, dhfr, ada, pac, hyg, mdr andhisD.

[0011] Transfected cells of the present invention are useful, aspopulations of transfected primary cells, transfected clonal cellstrains, transfected heterogenous cell strains, and as cell mixtures inwhich at least one representative cell of one of the three precedingcategories of transfected cells is present, as a delivery system fortreating an individual with a condition or disease which responds todelivery of EPO (e.g. anemia) or for preventing the development of sucha condition or disease. In the method of the present invention ofproviding EPO, transfected primary cells, clonal cell strains, orheterogenous cell strains, are administered to an individual in need ofEPO, in sufficient quantity and by an appropriate route, to deliver EPOto the systemic circulation at a physiologically relevant level. In asimilar manner, transfected cells of the present invention providinginsulinotropin are useful as populations of transfected primary cells,transfected clonal cell strains, transfected heterogenous cell strains,and as cell mixtures, as a delivery system for treating an individual inwhom insulin production, secretion or function is compromised or forinhibiting (totally or partially) glucagon secretion from the pancreas.A physiologically relevant level is one which either approximates thelevel at which the product is normally produced in the body or resultsin improvement of an abnormal or undesirable condition.

[0012] Clonal cell strains of transfected secondary cells (referred toas transfected clonal cell strains) expressing exogenous DNA encodingEPO (and, optionally, including a selectable marker gene) are producedby the method of the present invention. The present method includes thesteps of: 1) providing a population of primary cells, obtained from theindividual to whom the transfected primary cells will be administered orfrom another source; 2) introducing into the primary cells or intosecondary cells derived from primary cells a DNA construct whichincludes exogenous DNA encoding EPO and additional DNA sequencesnecessary for expression of EPO, thus producing transfected primary orsecondary cells; 3) maintaining transfected primary or secondary cellsunder conditions appropriate for their propagation; 4) identifying atransfected primary or secondary cell; and 5) producing a colony fromthe transfected primary or secondary cell identified in (4) bymaintaining it under appropriate culture conditions and for sufficienttime for its propagation, thereby producing a cell strain derived fromthe (founder) cell identified in (4). In one embodiment of the method,exogenous DNA encoding EPO is introduced into genomic DNA by homologousrecombination between DNA sequences present in the DNA construct used totransfect the recipient cells and the recipient cell's genomic DNA.Clonal cell strains of transfected secondary cells expressing exogenousDNA encoding insulinotropin (and, optionally, including a selectablemarker gene) are also produced by the present method.

[0013] In one embodiment of the present method of producing a clonalpopulation of transfected secondary cells, a cell suspension containingprimary or secondary cells is combined with exogenous DNA encoding EPOand DNA encoding a selectable marker, such as the bacterial neo gene.The two DNA sequences are present on the same DNA construct or on twoseparate DNA constructs. The resulting combination is subjected toelectroporation, generally at 250-300 volts with a capacitance of 960μFarads and an appropriate time constant (e.g., 14 to 20 msec) for cellsto take up the DNA construct. In an alternative embodiment,microinjection is used to introduce the DNA construct containingEPO-encoding DNA into primary or secondary cells. In either embodiment,introduction of the exogenous DNA results in production of transfectedprimary or secondary cells. Using the same approach, electroporation ormicroinjection is used to produce a clonal population of transfectedsecondary cells containing exogenous DNA encoding insulinotropin alone,or insulinotropin and a selectable marker.

[0014] In the method of producing heterogenous cell strains of thepresent invention, the same steps are carried out as described forproduction of a clonal cell strain, except that a single transfectedprimary or secondary cell is not isolated and used as the founder cell.Instead, two or more transfected primary or secondary cells are culturedto produce a heterogenous cell strain.

[0015] The subject invention also relates to a method of producingantibodies specific for EPO. In the method, transfected primary orsecondary cells expressing EPO are introduced into an animal recipient(e.g., rabbit, mouse, pig, dog, cat, goat, guinea pig, sheep, non-humanprimate). The animal recipient produces antibodies against the EPOexpressed, which may be the entire EPO protein antigen or a peptideencoded by a fragment of the intact EPO gene. Polyclonal sera isobtained from the animals. It is also possible to produce monoclonalantibodies through the use of transfected primary or secondary cells.Splenocytes are removed from an animal recipient of transfected primaryor secondary cells expressing EPO. The splenocytes are fused withmyeloma cells, using known methods, such as that of Koprowski et al.(U.S. Pat. No. 4,172,124) or Kohler et al., (Nature 256:495-497 (1975))to produce hybridoma cells which produce the desired anti-EPO monoclonalantibody. The polyclonal antisera and monoclonal antibodies produced canbe used for the same purposes (e.g., diagnostic, preventive, ortherapeutic purposes) as antibodies produced by other methods.Similarly, antibodies specific for insulinotropin can be produced by themethod of the present invention.

[0016] The present invention is particularly advantageous in treatinganemia and other conditions in which EPO production, utilization or bothis compromised in that it: 1) makes it possible for one gene therapytreatment, when necessary, to last a patient's lifetime; 2) allowsprecise dosing (the patient's cells continuously determine and deliverthe optimal dose of EPO based on physiologic demands, and the stablytransfected cell strains can be characterized extensively in vitro priorto implantation, leading to accurate predictions of long term functionin vivo); 3) is simple to apply in treating patients; 4) eliminatesissues concerning patient compliance (periodic administration of EPO isno longer necessary); and 5) reduces treatment costs (since thetherapeutic protein is synthesized by the patient's own cells,investment in costly protein production and purification facilities isunnecessary).

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic representation of plasmid pXEPO1. The solidblack arc represents the pUC12 backbone and the arrow denotes thedirection of transcription of the ampicillin resistance gene. Thestippled arc represents the mouse metallothionein promoter (pmMT1). Theunfilled arc interrupted by black boxes represents the humanerythropoietin EPO gene (the black boxes denote exons and the arrowindicates the direction hEPO transcription). The relative positions ofrestriction endonuclease recognition sites are indicated.

[0018]FIG. 2 is a schematic representation of plasmid pcDNEO. Thisplasmid has the neo gene from plasmid pSV2neo (a BamHI-BgIII fragment)inserted into the BamHI site of plasmid pcD; the amp and pBR322orisequences are from pBR322; the polyA, 19S splice junction, and earlypromoter sequences are from SV40.

[0019]FIG. 3 is a schematic representation of plasmid pXGH301. Thisplasmid contains both the human growth hormone (hGH) and neo resistancegenes. Arrows indicate the directions of transcription of the variousgenes. The positions of restriction endonuclease recognition sites, themouse metallothionein promoter (pMMT1), the amp resistance gene, and theSV40 early promoter (pSV40 early) are indicated.

[0020]FIG. 4 is a schematic representation of plasmid pE3neoEPO. Thepositions of the human erythropoietin gene and the neo and ampresistance genes are indicated. Arrows indicate the directions oftranscription of the various genes. pmMT1 denotes the mousemetallothionein promoter (driving hEPO expression) and pTK denotes theHerpes Simplex Virus thymidine kinase promoter (driving neo expression).The dotted regions of the map mark the positions of human HGPRTsequences. The relative positions of restriction endonucleaserecognition sites are indicated.

[0021]FIG. 5A shows results of Western blot analysis of hEPO secreted bynormal human fibroblasts cotransfected with pXEPO1 and pcDNEO. The leftpanel shows the Western analysis and the right panel shows a photographof the Coomassie blue stained gel. Lanes C, E, and M signify Controlsample (supernatant from untransfected human fibroblasts), Experimentalsample (supernatant from a clonal strain of human fibroblasts expressinghEPO), and marker lanes, respectively.

[0022]FIG. 5B shows results of Western blot analysis of hEPO secreted bynormal human fibroblasts cotransfected with pXEPO1 and pcDNEO.Supernatant from a clonal strain of human fibroblasts expressing hEPO(lane 1) was further analyzed for glycosylation by treatment withendoglycosidase-F (lane 2), neuraminidase (lane 3), and O-glycanase(lane 4).

[0023]FIG. 6A shows results of an assay to detect hEPO in the serum ofmice implanted with transfected rabbit fibroblasts expressing hEPO.

[0024]FIG. 6B shows hematocrit (HCT) levels in control mice and miceimplanted with transfected rabbit fibroblasts expressing hEPO.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to the use of geneticallyengineered cells to deliver a clinically useful or otherwise desirablesubstance to an individual in whom production of the substance isdesired (e.g., to prevent or treat a disease or condition in which theproduct is produced or functions at an unacceptable level). Inparticular, it relates to the use of genetically engineered cells todeliver EPO to the systemic circulation of an individual in need of EPO,resulting in an increase in mature red blood cell numbers, an increasein the oxygen-carrying potential of the blood, and an alleviation of thesymptoms of anemia. The present invention provides a means of deliveringEPO at physiologically relevant levels and on a continuous basis to anindividual. It further particularly relates to the use of geneticallyengineered cells to deliver insulinotropin to an individual in need ofinsulinotropin to stimulate insulin release, to increase insulinsensitivity in peripheral tissues, or to inhibit glucagon secretion fromthe pancreas.

[0026] As used herein, the term primary cell includes cells present in asuspension of cells isolated from a vertebrate tissue source (prior totheir being plated, i.e., attached to a tissue culture substrate such asa dish or flask), cells present in an explant derived from tissue, bothof the previous types of cells plated for the first time, and cellsuspensions derived from these plated cells. The term secondary cell orcell strain refers to cells at all subsequent steps in culturing. Thatis, the first time a plated primary cell is removed from the culturesubstrate and replated (passaged), it is referred to herein as asecondary cell, as are all cells in subsequent passages. Secondary cellsare cell strains which consist of secondary cells which have beenpassaged one or more times. A cell strain consists of secondary cellsthat: 1) have been passaged one or more times; 2) exhibit a finitenumber of mean population doublings in culture; 3) exhibit theproperties of contact-inhibited, anchorage dependent growth(anchorage-dependence does not apply to cells that are propagated insuspension culture); and 4) are not immortalized. A “clonal cell strain”is defined as a cell strain that is derived from a single founder cell.A “heterogenous cell strain” is defined as a cell strain that is derivedfrom two or more founder cells.

[0027] As described herein, primary or secondary cells of vertebrate,particularly mammalian, origin have been transfected with exogenous DNAencoding EPO and shown to produce the encoded EPO reproducibly, both invitro and in vivo, over extended periods of time. In addition, thetransfected primary and secondary cells have been shown to express EPOin vivo at physiologically relevant levels. The EPO expressed has beenshown to have the glycosylation pattern typical of EPO purified fromhuman urine or recombinant human EPO. This demonstration is in sharpcontrast to what one of skill in the art would predict, since, forexample, even experts in the field see the finite life span of normalsomatic cells and the inability to isolate or grow the relevanttransplantable cells as precluding their use for gene therapy unless thecells are genetically modified using retroviruses (Miller, A. D., Blood,76:271-278 (1990)). However, the transplantation of retrovirally treatedfibroblasts has been shown to provide, at best, only transient metabolicimprovements, and is seen to have serious limitations as a therapeuticsystem. In addition, until Applicants' work, this had not been done forEPO. Normal (non immortal) fibroblasts are characterized as being “muchmore difficult to transfect than continuous cell lines by using calciumphosphate precipitation techniques.” (Miller, A. D., Blood, 76:271-278(1990)). Furthermore, in considering non-retroviral techniques for genetherapy, it is typical of experts in the field to believe “ . . . theefficiency of gene delivery is dismal . . . A physician would have toobtain an impossible number of cells from patients to guarantee theappropriate alteration of the millions required for therapy.” (Verma, I.M. Scient. Amer., November 1990, pages 68-84).

[0028] Surprisingly, Applicants have been able to produce transfectedprimary and secondary cells which include exogenous DNA encoding EPO andexpress the exogenous DNA.

[0029] The transfected primary or secondary cells may also include DNAencoding a selectable marker which confers a selectable phenotype uponthem, facilitating their identification and isolation. Applicants havealso developed methods for producing transfected primary or secondarycells which stably express exogenous DNA encoding EPO, clonal cellstrains and heterogenous cell strains of such transfected cells, methodsof producing the clonal and heterogenous cell strains, and methods ofusing transfected cells expressing EPO to deliver the encoded product toan individual mammal at physiologically relevant levels. The constructsand methods are useful, for example, for treating an individual (human)whose EPO production and/or function is in need of being increased orenhanced [e.g., is compromised or less than normal, or normal but theindividual would benefit from enhancement, at least temporarily, of redblood cell production (e.g., during predialysis or dialysis therapy,during treatment of AIDS with AZT, after surgery, or duringchemotherapy)].

[0030] As also described herein, it is possible to transfect primary orsecondary cells of vertebrate, particularly mammalian, origin withexogenous DNA encoding insulinotropin and to use them to provideinsulinotropin to an individual in whom insulin production, functionand/or sensitivity is compromised.

[0031] Transfected Cells

[0032] Primary and secondary cells to be transfected in order to produceEPO or insulinotropin can be obtained from a variety of tissues andinclude all cell types which can be maintained and propagated inculture. For example, primary and secondary cells which can betransfected by the present method include fibroblasts, keratinocytes,epithelial cells (e.g., mammary epithelial cells, intestinal epithelialcells), endothelial cells, glial cells, neural cells, formed elements ofthe blood (e.g., lymphocytes, bone marrow cells), muscle cells, othersomatic cells which can be cultured, and precursors of these somaticcell types. Primary cells are preferably obtained from the individual towhom the transfected primary or secondary cells are administered.However, primary cells may be obtained from a donor (other than therecipient) of the same species or another species (e.g., mouse, rat,rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

[0033] Transfected primary and secondary cells can be produced, with orwithout phenotypic selection, as described herein, and shown to expressexogenous DNA encoding EPO or exogenous DNA encoding insulinotropin.

[0034] Exogenous DNA

[0035] Exogenous DNA incorporated into primary or secondary cells by thepresent method is DNA encoding the desired product (e.g., EPO orinsulinotropin), a functional or active portion, or a functionalequivalent of EPO or insulinotropin (a protein which has a differentamino acid sequence from that of EPO but has substantially the samebiological function as EPO, or a protein which has a different aminoacid-sequence from that of GLP-1 related peptides but functionsbiologically as insulinotropin). The DNA can be obtained from a sourcein which it occurs in nature or can be produced, using geneticengineering techniques or synthetic processes. The DNA encoding EPO orinsulinotropin will generally be DNA encoding the human product (i.e.,human EPO or human insulinotropin). In some cases, however, the DNA canbe DNA encoding EPO or insulinotropin of non-human origin (i.e., DNAobtained from a non-human source or DNA, produced recombinantly or bysynthetic methods, which encodes a non-human EPO or insulinotropin).

[0036] The DNA transfected into primary or secondary cells can encodeEPO alone or EPO and another product, such as a selectable marker tofacilitate selection and identification of transfected cells.Alternatively, the transfected DNA can encode insulinotropin alone orinsulinotropin and another product, such as a selectable marker. Aftertransfection into primary or secondary cells, the exogenous DNA isstably incorporated into the recipient cell's genome (along with theadditional sequences present in the DNA construct used), from which itis expressed or otherwise functions. Alternatively, the exogenous DNAmay exist episomally within the transfected primary or secondary cells.DNA encoding the desired product can be introduced into cells under thecontrol of an inducible promoter, with the result that cells produced oras introduced into an individual do not express the product but can beinduced to do so (i.e., production is induced after the transfectedcells are produced but before implantation or after implantation). DNAencoding the desired product can, of course, be introduced into cells insuch a manner that it is expressed upon introduction (i.e., withoutinduction).

[0037] Selectable Markers

[0038] A variety of selectable markers can be incorporated into primaryor secondary cells. For example, a selectable marker which confers aselectable phenotype such as drug resistance, nutritional auxotrophy,resistance to a cytotoxic agent or expression of a surface protein, canbe used. Selectable marker genes which can be used include neo, gpt,dhfr, ada, pac, hyg and hisD. The selectable phenotype conferred makesit possible to identify and isolate recipient primary or secondarycells.

[0039] DNA Constructs

[0040] DNA constructs, which include exogenous DNA encoding the desiredproduct (e.g., EPO, insulinotropin) and, optionally, DNA encoding aselectable marker, along with additional sequences necessary forexpression of the exogenous DNA in recipient primary or secondary cells,are used to transfect primary or secondary cells in which the protein(e.g., EPO, insulinotropin) is to be produced. Alternatively, infectiousvectors, such as retroviral, herpes, adenovirus, adenovirus-associated,mumps and poliovirus vectors, can be used for this purpose.

[0041] A DNA construct which includes the exogenous DNA encoding EPO andadditional sequences, such as sequences necessary for expression of EPO,can be used (e.g., plasmid pXEPO1; see FIG. 1). A DNA construct caninclude an inducible promoter which controls expression of the exogenousDNA, making inducible expression possible. Optionally, the DNA constructmay include a bacterial origin of replication and bacterial antibioticresistance markers, which allow for large-scale plasmid propagation inbacteria. A DNA construct which includes DNA encoding a selectablemarker, along with additional sequences, such as a promoter,polyadenylation site, and splice junctions, can be used to confer aselectable phenotype upon transfected primary or secondary cells (e.g.,plasmid pcDNEO). The two DNA constructs are co-transfected into primaryor secondary cells, using methods described herein. Alternatively, oneDNA construct which includes exogenous DNA encoding EPO, a selectablemarker gene and additional sequences (e.g., those necessary forexpression of the exogenous DNA and for expression of the selectablemarker gene) can be used. Such a DNA construct (pE3neoEPO) is describedin FIG. 4; it includes the EPO gene and the neo gene. Similarconstructs, which include exogenous DNA encoding insulinotropin andadditional sequences (e.g., sequences necessary for insulinotropinexpression) can be produced (e.g., plasmid pXGLP1; see Example 11).These constructs can also include DNA encoding a selectable marker, aswell as other sequences, such as a promoter, a polyadenylation site, andsplice junctions.

[0042] In those instances in which DNA is injected directly into anindividual, such as by injection intomusceles, the DNA constructincludes the exogenous DNA and regulatory sequences necessary andsufficient for expression of the encoded product (e.g., EPO) upon entryof the DNA construct into recipient cells.

[0043] Transfection of Primary or Secondary Cells and Production ofClonal or Heterogenous Cell Strains

[0044] Transfection of cells by the present method is carried out asfollows: vertebrate tissue is first obtained; this is carried out usingknown procedures, such as punch biopsy or other surgical methods ofobtaining a tissue source of the primary cell type of interest. Forexample, punch biopsy is used to obtain skin as a source of fibroblastsor keratinocytes. A mixture of primary cells is obtained from thetissue, using known methods, such as enzymatic digestion orexplantation. If enzymatic digestion is used, enzymes such ascollagenase, hyaluronidase, dispase, pronase, trypsin, elastase andchymotrypsin can be used.

[0045] The resulting primary cell mixture can be transfected directly orit can be cultured first, removed from the culture plate and resuspendedbefore transfection is carried out. Primary cells or secondary cells arecombined with exogenous DNA encoding EPO, to be stably integrated intotheir genomes and, optionally, DNA encoding a selectable marker, andtreated in order to accomplish transfection. The exogenous DNA andselectable marker-encoding DNA can each be present on a separateconstruct (e.g., pXEPO1 and pcDNEO, see FIGS. 1 and 2) or on a singleconstruct (e.g., pE3neoEPO, see FIG. 4). An appropriate quantity of DNAto ensure that at least one stably transfected cell containing andappropriately expressing exogenous DNA is produced. In general, 0.1 to500 μg DNA is used.

[0046] In one embodiment of the present method of producing transfectedprimary or secondary cells, transfection is effected by electroporation,as described in the Examples. Electroporation is carried out atappropriate voltage and capacitance (and corresponding time constant) toresult in entry of the DNA construct(s) into the primary or secondarycells. Electroporation can be carried out over a wide range of voltages(e.g., 50 to 2000 volts) and corresponding capacitance. As describedherein, electroporation is very efficient if carried out at anelectroporation voltage in the range of 250-300 volts and a capacitanceof 960 μFarads (see Examples 4, 5, 7 and 8). Total DNA of approximately0.1 to 500 μg is generally used. As described in the Examples, total DNAof 60 μg and voltage of 250-300 volts with capacitance of 960 μFaradsfor a time constant 14-20 of msec. has been used and shown to beefficient.

[0047] In another embodiment of the present method, primary or secondarycells are transfected using microinjection. See, for instance, Examples4 and 9. Alternatively, known methods such as calcium phosphateprecipitation, modified calcium phosphate precipitation and polybreneprecipitation, liposome fusion and receptor-mediated gene delivery canbe used to transfect cells. A stably, transfected cell is isolated andcultured and subcultivated, under culturing conditions and forsufficient time, to propagate the stably transfected secondary cells andproduce a clonal cell strain of transfected secondary cells.Alternatively, more than one transfected cell is cultured andsubculturated, resulting in production of a heterogenous cell strain.

[0048] Transfected primary or secondary cells undergo a sufficientnumber of doublings to produce either a clonal cell strain or aheterogenous cell strain of sufficient size to provide EPO to anindividual in effective amounts. In general, for example, 0.1 cm² ofskin is biopsied and assumed to contain 100,000 cells; one cell is usedto produce a clonal cell strain and undergoes approximately 27 doublingsto produce 100 million transfected secondary cells. If a heterogenouscell strain is to be produced from an original transfected population ofapproximately 100,000 cells, only 10 doublings are needed to produce 100million transfected cells.

[0049] The number of required cells in a transfected clonal orheterogenous cell strain is variable and depends on a variety offactors, which include but are not limited to, the use of thetransfected cells, the functional level of the exogenous DNA in thetransfected cells, the site of implantation of the transfected cells(for example, the number of cells that can be used is limited by theanatomical site of implantation), and the age, surface area, andclinical condition of the patient. To put these factors in perspective,to deliver therapeutic levels of EPO in an otherwise healthy 60 kgpatient with anemia, the number of cells needed is approximately thevolume of cells present on the very tip of the patient's thumb.

[0050] Episomal Expression of Exogenous DNA

[0051] DNA sequences that are present within the cell yet do notintegrate into the genome are referred to as episomes. Recombinantepisomes may be useful in at least three settings: 1) if a given celltype is incapable of stably integrating the exogenous DNA; 2) if a givencell type is adversely affected by the integration of DNA; and 3) if agiven cell type is capable of improved therapeutic function with anepisomal rather than integrated DNA.

[0052] Using the transfection and culturing approaches to gene therapydescribed in Examples 1 and 2, exogenous DNA encoding EPO, in the formof episomes can be introduced into vertebrate primary and secondarycells. Plasmid pE3neoEPO can be converted into such an episome by theaddition DNA sequences for the Epstein-Barr virus origin of replicationand nuclear antigen [Yates, J. L. Nature 319:780-7883 (1985)].Alternatively, vertebrate autonomously replicating sequences can beintroduced into the construct (Weidle, U. H. Gene 73(2):427-437 (1988).These and other episomally derived sequences can also be included in DNAconstructs without selectable markers, such as pXEPO1. The episomalexogenous DNA is then introduced into primary or secondary vertebratecells as described in this application (if a selective marker isincluded in the episome, a selective agent is used to treat thetransfected cells). Similarly, episomal expression of DNA encodinginsulinotropin can be accomplished in vertebrate primary or secondarycells, using the same approach described above with reference to EPO.

[0053] Implantation of Clonal Cell Strain or Heterogenous Cell Strain ofTransfected Secondary Cells

[0054] The transfected cells produced as described above are introducedinto an individual to whom EPO is to be delivered, using known methods.The clonal cell strain or heterogenous cell strain is introduced into anindividual, using known methods, using various routes of administrationand at various sites (e.g., renal subcapsular, subcutaneous, centralnervous system (including intrathecal), intravascular, intrahepatic,intrasplanchnic, intraperitoneal (including intraomental), orintramuscular implantation)]. Once implanted in the individual, thetransfected cells produce EPO encoded by the exogenous DNA. For example,an individual who has been diagnosed as anemic is a candidate for a genetherapy cure. The patient has a small skin biopsy performed; this is asimple procedure which can be performed on an out-patient basis. Thepiece of skin, approximately 0.1 cm², is taken, for example, from underthe arm and requires about one minute to remove. The sample isprocessed, resulting in isolation of the patient's cells (in this case,fibroblasts) and genetically engineered to produce EPO. Based on theage, weight, and clinical condition of the patient, the required numberof cells is grown in large-scale culture. The entire process usuallyrequires 4-6 weeks and, at the end of that time, the appropriate numberof genetically-engineered cells is introduced into the individual (e.g.,by injecting them back under the patient's skin). The patient is nowcapable of producing his or her own EPO or additional EPO.

[0055] Transfected cells, produced as described above, which containinsulinotropin-encoding DNA are delivered into an individual in whominsulin production, secretion, function and/or sensitivity iscompromised. They are introduced into the individual by known methodsand at various sites of administration (e.g., renal, subcapsular,subcutaneous, central nervous system (including intrathecal),intravascular, intrahepatic, intrasplanchnic, intraperitoneal (includingintraomental) or intramuscular implantation). Once implanted in theindividual, the transfected cells produce insulinotropin encoded by theexogenous DNA. For example, an individual in whom insulin production,secretion or sensitivity is impaired can receive therapy or preventivetreatment through the implantation of transfected cells expressingexogenous DNA encoding insulinotropin produced as described herein. Thecells to be genetically engineered are obtained as described above forEPO, processed in a similar manner to produce sufficient numbers ofcells, and introduced back into the individual.

[0056] As this example suggests, the cells used will generally bepatient-specific genetically-engineered cells. It is possible, however,to obtain cells from another individual of the same species or from adifferent species. Use of such cells might require administration of animmunosuppressant, alteration of histocompatibility antigens, or use ofa barrier device to prevent rejection of the implanted cells.

[0057] In one embodiment, a barrier device is used to prevent rejectionof implanted cells obtained from a source other than the recipient(e.g., from another human or from a non-human mammal such as a cow, dog,pig, goat, sheep or rodent). In this embodiment, transfected cells ofthe present invention are placed within the barrier device, which ismade of a material (e.g., a membrane such as Amicon XM-50) which permitsthe product encoded by the exogenous DNA to pass into the recipient'scirculation or tissues but prevents contact between the cells and therecipient's immune system and thus prevents an immune response to (andpossible rejection of) the cells by the recipient. Alternatively, DNAencoding EPO or insulinotropin can be introduced into an individual bydirect injection, such as into muscle or other appropriate site. In thisembodiment, the DNA construct includes exogenous DNA encoding thetherapeutic product (e.g., EPO, insulinotropin) and sufficientregulatory sequences for expression of the exogenous DNA in recipientcells. After injection into the individual, the DNA construct is takenup by some of the recipient cells. The DNA can be injected alone or in aformulation which includes a physiologically compatible carrier (e.g., aphysiological buffer) and, optionally, other components, such as agentswhich allow more efficient entry of the DNA construct into cells,stabilize the DNA or protect the DNA from degradation.

[0058] Uses of Transfected Primary and Secondary Cells and Cell Strains

[0059] Transfected primary or secondary cells or cell strains have wideapplicability as a vehicle or delivery system for EPO. The transfectedprimary or secondary cells of the present invention can be used toadminister EPO, which is presently administered by intravenousinjection. When transfected primary or secondary cells are used, thereis no need for extensive purification of the polypeptide before it isadministered to an individual, as is generally necessary with anisolated polypeptide. In addition, transfected primary or secondarycells of the present invention produce the therapeutic product as itwould normally be produced.

[0060] An advantage to the use of transfected primary or secondary cellsof the present invention is that by controlling the number of cellsintroduced into an individual, one can control the amount of EPO. Inaddition, in some cases, it is possible to remove the transfected cellsif there is no longer a need for the product. A further advantage oftreatment by use of transfected primary or secondary cells of thepresent invention is that production can be regulated, such as throughthe administration of zinc, steroids or an agent which affectstranscription of the EPO-encoding DNA.

[0061] Glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 1derivatives (GLP-1 derivatives) are additional molecules that can bedelivered therapeutically using the in vivo protein production anddelivery system described in the present invention. GLP-1 derivativesinclude truncated derivatives GLP-1(7-37), GLP-1(7-36), GLP-1(7-35)GLP-1(7-34) and other truncated carboxy-terminal amidated derivativesand derivatives of GLP-1 which have amino acid substitutions, deletions,additions or other alterations (e.g., addition of a non-amino acidcomponent) which result in biological activity or stability in the bloodwhich is substantially the same as that of a truncated GLP-1 derivativeor enhanced biological activity or stability in the blood (greater thanthat of a truncated GLP-1 derivative). As used herein, the term GLP-1derivative includes all of the above-described molecules. The term GLP-1related peptide, as used herein, includes GLP-1 and GLP-1 derivatives.GLP-1 derivatives, also known as insulinotropins or incretins, arenormally secreted into the circulation by cells in the gastrointestinaltract. In vivo studies have demonstrated that these peptides function tostimulate insulin secretion and inhibit glucagon secretion from theendocrine pancreas, as well as increase insulin sensitivity inperipheral tissues [Goke, R. et al. (1991) Eur. J. Clin. Inv.21:135-144; Gutniak, M. et al. (1992) New Engl. J. Med. 326:1316-1322].Patients with non-insulin dependent diabetes mellitus (NIDDM) are oftentreated with high levels of insulin to compensate for their decreasedinsulin sensitivity. Thus, the stimulation of insulin release and theincrease in insulin sensitivity by GLP-1 derivatives would be beneficialfor NIDDM patients. Of particular importance is the fact that theinsulinotropin-induced stimulation of insulin secretion is stronglydependent on glucose levels, suggesting that these peptides act inresponse to increases in blood glucose in vivo to potentiate insulinrelease and, ultimately, lower blood glucose.

[0062] The present invention will now be illustrated by the followingexamples, which are not intended to be limiting in any way.

EXAMPLES Example 1 Isolation Of Fibroblasts

[0063] a. Source of Fibroblasts.

[0064] Human fibroblasts can be obtained from a variety of tissues,including biopsy specimens derived from liver, kidney, lung and skin.The procedures presented here are optimized for the isolation of skinfibroblasts, which are readily obtained from individuals of any age withminimal discomfort and risk (embryonic and fetal fibroblasts may beisolated using this protocol as well). Minor modifications to theprotocol can be made if the isolation of fibroblasts from other tissuesis desired.

[0065] Human skin is obtained following circumcision or punch biopsy.The specimen consists of three major components: the epidermal anddermal layers of the skin itself, and a fascial layer that adheres tothe dermal layer. Fibroblasts can be isolated from either the dermal orfascial layers.

[0066] b. Isolation of Human Fascial Fibroblasts.

[0067] Approximately 3 cm² tissue is placed into approximately 10 ml ofwash solution (Hank's Balanced Salt Solution containing 100 units/mlpenicillin G, 100 μg/ml streptomycin sulfate, and 0.5 μg/ml Fungisone)and subjected to gentle agitation for a total of three 10-minute washesat room temperature. The tissue is then transferred to a 100 mm tissueculture dish containing 10 ml digestion solution (wash solutioncontaining 0.1 units/ml collagenase A, 2.4 units/ml grade II Dispase).

[0068] Under a dissecting microscope, the skin is adjusted such that theepidermis is facing down. The fascial tissue is separated from thedermal and epidermal tissue by blunt dissection. The fascial tissue isthen cut into small fragments (less than 1 mm²) and incubated on arotating platform for 30 min at 37° C. The enzyme/cell suspension isremoved and saved, an additional 10 cc of digestion solution is added tothe remaining fragments of tissue, and the tissue is reincubated for 30min at 37° C. The enzyme/cell suspensions are pooled, passed through a15-gauge needle several times, and passed through a Cellector Sieve(Sigma) fitted with a 150-mesh screen. The cell suspension iscentrifuged at 1100 rpm for 15 min at room temperature. The supernatantis aspirated and the disaggregated cells resuspended in 10 ml ofnutrient medium (see below). Fibroblast cultures are initiated on tissueculture treated flasks (Corning) at a density of approximately 40,000cells/cm².

[0069] c. Isolation of Human Dermal Fibroblasts.

[0070] Fascia is removed from skin biopsy or circumcision specimen asdescribed above and the skin is cut into small fragments less than 0.5cm². The tissue is incubated with 0.25% trypsin for 60 min at 37° C.(alternatively, the tissue can be incubated in trypsin for 18 hrs at 4°C.). Under the dissecting microscope, the dermis and epidermis areseparated. Dermal fibroblasts are then isolated as described above forfascial fibroblasts.

[0071] d. Isolation of Rabbit Fibroblasts.

[0072] The procedure is essentially as described above. Skin should beremoved from areas that have been shaved and washed with a germicidialsolution and surgically prepared using accepted procedures.

Example 2 Culturing of Fibroblasts

[0073] a. Culturing of Human Fibroblasts.

[0074] When confluent, the primary culture is trypsinized using standardmethods and seeded at approximately 10,000 cells/cm². The cells arecultured at 37° C. in humidified air containing 5% CO₂. Human fibroblastnutrient medium (containing DMEM, high glucose with sodium pyruvate,10-15% calf serum, 20 mM HEPES, 20 mM L-glutamine, 50 units/mlpenicillin G, and 10 μg/ml streptomycin sulfate) is changed twiceweekly.

[0075] b. Culturing of Rabbit Fibroblasts.

[0076] The cells are trypsinized and cultured as described for humanfibroblasts. Rabbit fibroblast nutrient medium consists of a 1:1solution of MCDB-110 (Sigma) with 20% calf serum and conditioned medium.Conditioned medium is essentially human fibroblast nutrient medium (with15% calf serum) removed from rabbit fibroblasts grown in mass culturefor 2-3 days.

Example 3 Construction of a Plasmid (pXEPO1) Containing the HumanErythropoietin Gene Under the Control of the Mouse MetallothioneinPromoter

[0077] The expression plasmid pXEPO1 has the hEPO gene under thetranscriptional control of the mouse metallothionein (mMT) promoter.pXEPO1 is constructed as follows: Plasmid pUC19 (ATCC #37254) isdigested with KpnI and BamHI and ligated to a 0.7 kb KpnI-BgIII fragmentcontaining the mouse metallothionein promoter [Hamer, D. H. and Walling,M., J. Mol. Appl. Gen., 1:273-288 (1982). This fragment can also beisolated by known methods from mouse genomic DNA using PCR primersdesigned from analysis of mMT sequences available from Genbank; i.e.MUSMTI, MUSMTIP, MUSMTIPRM]. The resulting clone is designated pXQM2.

[0078] The hEPO gene was isolated by from a bacteriophage lambda clonecontaining the entire hEPO gene. This bacteriophage was isolated byscreening a human Sau3A-partial genomic DNA library (Stratagene)constructed in the lambda vector LAMBDA DASH with 0.77 kb fragment ofthe human gene. This 0.77 kb fragment was amplified from human genomicDNA using the primers shown below in the polymerase chain reaction(PCR).

[0079] Human EPO PCR Primers: Oligo hEPO-1: 5′GTTTGCTCAGCTTGGTGCTTG(Seq. ID No. 1)

[0080] (positions 2214-2234 in the Genbank HUMERPA sequence) OligohEPO-2: 5′TCAAGTTGGCCCTGTGACAT (Seq. ID No. 2)

[0081] (positions 2986-2967 in the Genbank HUMERPA sequence)

[0082] The amplified fragment, encompassing exons 4 and 5 of the humanEPO gene, was radiolabelled and used to screen the human genomic DNAlibrary. Phage with a 5.4 kb HindIII-BamHI fragment containing theentire human EPO gene were assumed to contain the entire gene, based onpublished DNA sequence and restriction enzyme mapping data [Lin, F-K.,et al., Proc. Natl. Acad. Sci. USA, 82:7580-7584 (1985)].

[0083] A 4.8 kb BstEII-BamHI fragment (BstEII site is at position 580 inGenbank HUMERPA sequence; the BamHI site is 4.8 kb 3′ of this site,outside of the sequenced region) was isolated from the bacteriophageclone. The purified fragment is made blunt-ended by treatment with theKlenow fragment of E. coli DNA polymerase and ligated to HincII digestedpXQM2, which cuts in the pUC19-derived polylinker adjacent to the 3′side of the subcloned mMT promoter. One orientation, in which theablated BstEII site is proximal to the mMT promoter, was identified byrestriction mapping and designated PXEPO1 (FIG. 1).

Example 4 Transfection of Primary and Secondary Fibroblasts WithExogenous DNA and a Selectable Marker Gene by Electroporation andMicroinjection

[0084] To prepare cells for electroporation, exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation as described above. The supernatant is aspirated and thepellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HPO₄, 6 mM dextrose). The cellsare recentrifuged, the supernatant aspirated, and the cells resuspendedin electroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0085] Supercoiled plasmid DNA is added to a sterile cuvette with a 0.4cm electrode gap (Bio-Rad). The final DNA concentration is generally atleast 120 μg/ml. 0.5 ml of the cell suspension (containing approximately1.5×10⁶ cells) is then added to the cuvette, and the cell suspension andDNA solutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0086] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (as above with 15% calf serum) in a 10cm dish and incubated as described above. The following day, the mediais aspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hrs. Subculture of cells to determine cloning efficiencyand to select for G418-resistant colonies is performed the followingday. Cells are trypsinized, counted and plated; typically, fibroblastsare plated at 103 cells/10 cm dish for the determination of cloningefficiency and at 1-2×10⁴ cells/10 cm dish for G418 selection.

[0087] Human fibroblasts are selected for G418 resistance in mediumconsisting of 300-400 μg/ml G418 (Geneticin, disulfate salt with apotency of approximately 50%; Gibco) in fibroblasts nutrient media (with15% calf serum). Cloning efficiency is determined in the absence ofG418. The plated cells are incubated for 12-14 days, at which timecolonies are fixed with formalin, stained with crystal violet andcounted (for cloning efficiency plates) or isolated using cloningcylinders (for G418 plates). Electroporation and selection of rabbitfibroblasts is performed essentially as described for human fibroblasts,with the exception of the nutrient media used. Rabbit fibroblasts areselected for G418 resistance in medium containing 1 mg/ml G418.

[0088] Fibroblasts were isolated from freshly excised human foreskins.Cultures were seeded at 50,000 cells/cm² in DMEM+10% calf serum. Whencultures became confluent fibroblasts were harvested by trypsinizationand transfected by electroporation. Electroporation conditions wereevaluated by transfection with the plasmid pcDNEO. A representativeelectroporation experiment using near optimal conditions (60 μg ofplasmid pcDNEO at an electroporation voltage of 250 volts and acapacitance setting of 960 μFarads) resulted in one G418^(r) coloney per588 treated cells (0.17% of all cells treated), or one G418^(r) colonyper 71 clonable cells (1.4%).

[0089] When nine separate electroporation experiments at near optimalconditions (60 μg of plasmid pcDNEO at an electroporation voltage of 300volts and a capacitance setting of 960 μFarads) were performed, anaverage of one G418^(r) colony per 1,899 treated cells (0.05%) wasobserved, with a range of {fraction (1/882)} to {fraction (1/7,500)}treated cells. This corresponds to an average of one G418^(r) colony per38 clonable cells (2.6%).

[0090] Low passage primary human fibroblasts were converted to hGHexpressing cells by co-transfection with plasmids pcDNEO and pXGH5[Selden, R. F. et al., Mol. Cell. Biol., 6:3173-3179 (1986)]. Typically,60 μg of an equimolar mixture of the two plasmids were transfected atnear optimal conditions (electroporation voltage of 300 volts and acapacitance setting of 960 μFarads). The results of such an experimentresulted in one G418^(r) colony per 14,705 treated cells.

[0091] hGH expression data for these and other cells isolated underidentical transfection conditions are summarized below. Ultimately, 98%of all G418^(r) colonies could be expanded to generate mass cultures.Number of G418^(r) Clones 154 Analyzed Number of G418^(r)/hGH 65Expressing Clones Average hGH Expression 2.3 μg hGH/10⁶ Cells/24 hrLevel Maximum hGH Expression 23.0 μg hGH/10⁶ Cells/24 hr Level

[0092] Stable transfectants also have been generated by electroporationof primary or secondary human fibroblasts with pXGH301, a DNA constructin which the neo and hGH genes are present on the same plasmid molecule(Example 3). For example, 1.5×10⁶ cells were electroporated with 60 μgpXGH301 at 300 volts and 960 μFarads. G418 resistant colonies wereisolated from transfected secondary fibroblasts at a frequency of 652G418 resistant colonies per 1.5×10⁶ treated cells (1 per 2299 treatedcells). Approximately 59% of these colonies express hGH.

[0093] Primary and secondary human fibroblasts can also be transfectedby direct injection of DNA into cell nuclei. The ability of primary andsecondary human foreskin fibroblasts to be stably transfected by thismethod has not been previously reported. The 8 kb HindIII fragment fromplasmid RV6.9h (Zheng, H. et al., Proc. Natl. Acad. Sci. USA 88:188067-8071 (1991)) was purified by gel electrophoresis and passagethrough an anion exchange column (QIAGEN Inc.). DNA at (10 μg/ml) wasinjected into primary or secondary human foreskin fibroblasts using 0.1μm outer diameter glass needles. 41 G₄₁₈ ^(r) clones were isolated afterinjection of 2,000 cells (1 in 49 starting cells).

[0094] hGH expressing clones were also generated by microinjection.Plasmid pXGH301 (FIG. 3) was linearized by ScaI digestion (which cutsonce within the amp^(r) gene in the pUC12 backbone), purified by passagethrough an anion exchange column (QIAGEN Inc.), and injected intosecondary human foreskin fibroblasts using 0.1 μm outer diameter glassneedles. Several DNA concentrations were used, ranging from 2.5-20 μgpXGH301/ml. Twenty G418 resistant clones were isolated aftermicroinjection into 2,100 cells (1 in 105 starting cells). The fractionof G418^(r) cells, is approximately 1% of all cells treated. Nine of 10clones analyzed were expressing hGH, with average hGH expression being0.6 μg/10⁶ cells/24 hr for clones isolated in this experiment, and 3clones were expanded for studying long-term expression of hGH. All 3were expressing hGH stably, with hGH still being produced through 33,44, and 73 mpd for the 3 strains, respectively.

Example 5 In vitro hEPO Production by Transfected Secondary Human andRabbit Skin Fibroblasts

[0095] 1. Human Skin Fibroblasts

[0096] Fibroblasts were isolated from freshly excised human skinfibroblasts and cultured in DMEM+15% calf serum.

[0097] Electroporation (250 volts, 960 μfarads) with 60 μg of anequimolar mixture of pcDNEO and pXEPO1 was performed on passage 1 cellsand treated cells were selected in G418-containing medium (300 μg/mlG418). Colonies were isolated and expanded using standard methods. Dataderived from an analysis of fifty-six individual clones is shown inTable 1 below. Cells were maintained in G418 (300 μg/ml G418) inDMEM+15% calf serum and subcultured at a seeding density of 10,000cells/cm². Culture medium was changed 24 hr prior to harvesting thecells for passaging. At the time of passage, an aliquot of the culturemedium was removed for hEPO assay and the cells were then harvested,counted, and reseeded. hEPO concentration in the medium was determinedusing a commercially available ELISA (R & D Systems). hEPO levels areexpressed as mU/10⁶ cells/24 hr., and expression levels ranged from 69to 55,591 mU/10⁶ cells/24 hr. 19% of all G418-resistant coloniesexpressed detectable levels of hEPO.

Table 1 hEPO Expression in Fifty-six Independent Secondary HumanFibroblast Clones Isolated by Co-transfection With pcDNEO and pXEPO1

[0098] HEPO Expression Level (mU/10⁶ cells/24 hr) Number of Clones <1,000 10  1,000-10,000 28 10,000-50,000 17 >50,000  1

[0099] Clonally derived human fibroblasts isolated by co-transfectionwith pcDneo and pXEPO1 were analyzed for the glycosylation state ofsecreted hEPO. Media was collected from hEPO producing cells andimmunoprecipitated with a mouse monoclonal antibody (GenzymeCorporation) specific for human erythropoietin. The immunoprecipitatedmaterial was subject to electrophoresis on a 12.5% polyacrylamide geland transferred to a PVDF membrane (Millipore). The membrane was probedwith the same anti-hEPO monoclonal antibody used for immunoprecipitationand was subsequently treated with an HRP-conjugated sheep anti-mouse IgGantisera (Cappel), followed by luminescent detection (ECL Westernblotting detection kit; Amersham) to visualize hEPO through theproduction of a fluorescent product.

[0100] As shown in FIG. 5A, a molecule with a molecular mass ofapproximately 34 kd reacts with an antibody specific for humanerythropoietin. This is the expected size for naturally occurring, fullyglycosylated human erythropoietin.

[0101] hEPO produced by transfected human fibroblast clones was furtheranalyzed to determine if the secreted material had both N- and O-linkedglycosylation characteristic of natural human erythropoietin isolatedfrom urine or recombinant hEPO produced by chinese hamster ovary cells.FIG. 5B shows a Western blot of the untreated cell supernatant (lane 1),the supernatant treated with endoglycosidase-F [(New England Nuclear);lane 2], the supernatant treated with neuraminidase [Genzyme); (lane3)], and the supernatant treated with O-glycanase [(Genzyme); (lane 4)].Treatment with endoglycosidase-F results in a shift in molecular weightfrom 34 kd to approximately 27 kd. Treatment with neuraminidase resultsin a barely detectable shift in band position, while treatment withO-glycanase further shifts the size of the immunoreactive band down toapproximately 18.5 kd. These results are indistinguishable from thoseobtained with natural human erythropoietin isolated from urine orrecombinant hEPO produced by Chinese hamster ovary cells (Browne, J. K.et al., Cold Spring Harbor Symp. Quant. Biol. 51:693-702 (1986)).

[0102] 2. Rabbit Fibroblasts

[0103] Fibroblasts were isolated from freshly excised rabbit skin andcultured in DMEM 10% calf serum. Electroporation (250 volts, 960μFarads) with 60 μg of an equimolar mixture of pcDNEO and pXEPO1 wasperformed and treated cells were selected in G418-containing rabbitfibroblast growth medium (1 mg/ml G418; Example 2). Colonies wereisolated and expanded using standard methods, and the resultingsecondary cell strains were analyzed for hEPO expression. Data derivedfrom forty-nine independent rabbit fibroblast clones is shown in Table 2below. Expression levels in these clones ranged from 43 to 2,900,000mU/10⁶ cells/24 hr., and 72% of all G418-resistant clones expresseddetectable levels of hEPO. TABLE 2 hEPO EXPRESSION IN FORTY-NINEINDEPENDENT SECONDARY RABBIT FIBROBLAST CLONES ISOLATED BYCO-TRANSFECTION WITH pcDNEO AND pEEPO hEPO Expression Level (mU/10⁶cells/24 hr) Number of Clones  <1,000 1  1,000-10,000 3 10,000-50,000 7 50,000-500,000 19  >500,000 19 

Example 6 Construction of a Plasmid Containing Both the Human EPO Geneand the Neomycin Resistance Gene

[0104] A 6.9 kb HindIII fragment extending from positions 11,960-18,869in the HPRT sequence [Genbank entry HUMHPRTB; Edwards, A. et al.,Genomics, 6:593-608 (1990)] and including exons 2 and 3 of the HPRTgene, is subcloned into the HindIII site of pUC12. The resulting cloneis cleaved at the unique Xhol site in exon 3 of the HPRT gene fragmentand the 1.1 kb SaII-XhoI fragment containing the neo gene from pMClNEO(Stratagene) is inserted, disrupting the coding sequence of exon 3. Oneorientation, with the direction of neo transcription opposite that ofHPRT transcription was chosen and designated pE3Neo. pE3neo has a uniqueXhoI site at the junction of HPRT sequences and the 5′ side of the neogene. pE3neo is cut with XhoI and made blunt-ended by treatment with theKlenow fragment of E. coli DNA polymerase.

[0105] To insert the hEPO gene into the neo selection plasmid pE3Neo, a5.1 kb EcoRI-HindIII fragment was isolated from plasmid pXEPO1 (Example3; FIG. 1). The EcoRI site is located adjacent to the 5′ side of the mMTpromoter, and the HindIII site is located 5.1 kb away, 3′ to the hEPOcoding region. The purified Fragment is made blunt-ended by treatmentwith Klenow fragment of E. coli DNA polymerase and ligated to the XhoIdigested and blunt-ended pE3neo fragment described above. Aftertransformation into E. coli, a plasmid with one copy of the mMT-hEPOfragment inserted into pE3neo was identified by restriction enzymeanalysis in which the hEPO gene is transcribed in the same orientationas the adjacent neo gene. This plasmid was designated pE3neoEPO. Inaddition to allowing direct selection of hEPO expressing G418^(r)clones, this fragment may also be used in gene targeting to direct theintegration of the hEPO gene to the human HPRT locus.

Example 7 Isolation of Human Fibroblast Clones Expressing hEPO Gene anda Selectable Marker (pE3neoEPO)

[0106] Fibroblasts were isolated from freshly excised human skinfibroblasts and cultured in DMEM+15% calf serum. Electroporation (250volts, 960 μFarads) with 60 μg of supercoiled pE3neoEPO was performed onpassage 1 cells and treated cells were selected in G418-containingmedium (300 μg/ml G418). Colonies were isolated and expanded usingstandard methods. Data derived from an analysis of twenty-six individualclones is shown in Table 3 below. Cells were maintained in G418 (300μg/ml G418) in DMEM+15% calf serum and subcultured at a seeding densityof 10,000 cells/cm². Culture medium was changed 24 hr prior toharvesting the cells for passaging. At the time of passage an aliquot ofthe culture medium was removed for hEPO assay and the cells were thenharvested, counted, and reseeded. hEPO concentration in the medium wasdetermined using a commercially available ELISA (R and D Systems). hEPOlevels are expressed as mU hEPO/10⁶ cells/24 hr, and expression levelsranged from 240 to 961,620 mU/10⁶ cells/24 hr. 89% of all G418-resistantclones expressed detectable levels of hEPO. TABLE 3 hEPO EXPRESSION INTWENTY-SIX INDEPENDENT SECONDARY HUMAN FIBROBLAST CLONES ISOLATED BYTRANSFECTION WITH pE3neo-EPO hEPO Expression Level (mU/10⁶ cells/24 hr)Number of Clones  <1,000 2  1,000-10,000 2 10,000-50,000 9 50,000-500,000 12  >500,000 1

[0107] hEPO expressing human fibroblast clones are also isolated byelectroporation with 60 μg of HindIII digested pE3neoEPO. hEPOexpressing rabbit fibroblast clones are isolated using plasmid pE3neoEPOunder identical transfection conditions, with the exception that rabbitfibroblast clones are selected in rabbit fibroblast growth medium(Example 2) containing 1 mg/ml G418.

Example 8 Isolation of Transfectants in the Absence of Selection

[0108] The high frequency of transfection in human fibroblasts (greaterthan 1% stable transfectant per clonable cell; Example 4) indicates thatit should be possible to isolate cell clones that have stablyincorporated exogenous DNA without the use of a selective agent. Stabletransfection of primary fibroblasts with the plasmid pXEPO1 shouldrender recipient fibroblasts capable of secreting human erythropoietininto the surrounding medium. Therefore, an ELISA for hEPO (or for anyexpressed protein of therapeutic interest) can be used as a simple andrapid screen for transfectants. Alternatively, it should be possible todetermine the true frequency of stable integration of exogenous DNAusing a screening method such as PCR which does not necessarily rely onexpression of transfected DNA.

[0109] 1. Primary Human Fibroblasts

[0110] Approximately 2.0×10⁶ human cells that were freshly dissociatedfrom tissue are electroporated with 60 μg of pXEPO1 at 300 volts, 960μFarads. Cells are plated immediately in a 100 mm tissue culture dishcontaining 10 ml of prewarmed medium and incubated at 37° C. in ahumidified 5% CO₂ atmosphere. Two days following transfection, 5×10³cells are subcultured into a 24 well cloning plate (Bellco Glass Co.).Each well of the 24 well plate contained 16 smaller wells (384wells/plate). Eight days after plating into the 24 large wells, media isscreened for hEPO expression via ELISA. A second, confirming assay, isdone in which media from wells exhibiting hEPO expression is aspiratedout, replaced with fresh media and assayed for hEPO 24 hours later.Colony counts at this stage should reveal approximately 10 colonies perlarge well.

[0111] Individual colonies in each of the 16 small wells within one ofthe hEPO-positive larger wells are trypsinized and transferred to wellsof a 96 well plate. Three days later each of those wells are assayed forhEPO expression. Cells from hEPO positive cells are expanded for furtherstudy. This experiment may also be performed using secondary humanforeskin fibroblasts.

[0112] 2. Primary Rabbit Fibroblasts

[0113] Passage 1 rabbit skin cells were transfected with pXEPO1. Theelectroporation conditions were identical to the human tissueelectroporation described above. 1×10³ cells are subcultured into a 384well plate. Seven days later hGH assays are performed on media from eachof the 24 large wells. Cells in each of the small wells in hEPO-positivelarge wells are trypsinized and transferred to wells of a 96 well plate.Three days later each of these wells are assayed for hEPO expression.Cells from hEPO positive cells are expanded for further study. Thisexperiment may also be performed using secondary rabbit skinfibroblasts.

Example 9 Stable Transfection of Primary Human Fibroblasts byMicroinjection

[0114] Direct injection of DNA into cell nuclei is another method forstably transfecting cells. The ability of primary and secondary humanforeskin fibroblasts to be stably transfected by this method isdescribed in Example 4, but has not been previously reported in theliterature. The 13.1 kb HindIII fragment from plasmid pE3neoEPO ispurified by gel electrophoresis and passed through an anion exchangecolumn (QIAGEN Inc.). This fragment contains the human EPO and bacterialneo genes, flanked on both sides with human HPRT sequences. DNA at (10μg/ml) is injected into primary or secondary human foreskin fibroblastsusing 0.1 μm diameter glass needles. G₄₁₈ ^(r) clones are isolatedapproximately 12-14 days after injection. Alternatively, the totalHindIII digest of pE3neoEPO, as well as linearized or supercoiledpE3neoEPO may be injected to isolate hEPO expressing cells.

Example 10 Expression of Biologically Active Human Erythropoietin inMice

[0115] The mouse provides a valuable system to study implants ofgenetically engineered cells for their ability to delivertherapeutically useful proteins to an animal's general circulation. Therelative immune-incompetence of nude mice allow xenogeneic implants toretain biologic function and may allow certain primary and secondaryrabbit fibroblasts to survive in vivo for extended periods.

[0116] For implantation of cells into the subrenal capsule, mice aregiven intraperitoneal injection of Avertin at a dose of 0.0175 ml/g bodyweight. The kidney (generally the left kidney) is approached through an8-10 mm incision made approximately 3 mm below the rib cage. The skin,abdominal musculature, peritoneum, and peri-renal fascia are retractedto expose the kidney. A small forcep is used to pull the kidney out ofthe abdominal cavity. A 27-gauge hypodermic needle is used to make asmall opening in the renal capsule. Using a 20-gauge I.V. catheter,cells to be implanted (typically 3 million cells in a volume of 5-10 μl)are withdrawn into a 1 ml syringe and slowly ejected under the renalcapsule. Care is taken to ensure that the cells are released distal tothe opening in the renal capsule. The incision is closed with one staplethrough the musculature and the skin. Blood is collected after placingthe mouse in a large beaker containing methoxyflurane until lightanesthesia is achieved. The tip of a Pasteur pipette is placed betweenthe eye and the periorbital space to collect blood from the orbitalsinus. Serum hEPO levels are determined using a commercially availablekit (R and D Systems). An aliquot of blood is also drawn into EDTAcoated capillary tubes (Statspin, Norwood, MA) for determination ofhematocrit levels.

[0117] A clonal strain of rabbit skin fibroblasts was isolated by themethods described in Example 5. One clone, designated RF115-D4, wasdetermined to be stably transfected with the human EPO gene andexpressed approx-6 imately 786,000 mU hEPO/10⁶ cells/24 hr. Threemillion cells were implanted into the subrenal capsule in each of sixnude mice. Approximately 400 μl of blood was drawn on days 1 and 7 afterimplantation and on every other day thereafter until day 21. During thistime an equal volume of saline solution was injected after bleeding toprevent hypotonic shock. Blood was drawn weekly thereafter until day 63.An identical bleeding schedule was used on ten mice that had no cellsimplanted. FIG. 6A shows the effect of these treatments on bloodhematocrit (HCT), a commonly used indicator of red blood cell number, inimplanted and control animals. In control animals, HCT dropsdramatically by day 7, followed by a return to approximately normallevels by day 15. In contrast, animals receiving implants of the hEPOexpressing cells showed elevated HCT levels by day 7. HCT remainedelevated through day 63, reaching a peak of 64%, or 1.4 times higherthan the day 1 level of 45%, on day 35 after implantation. As shown inFIG. 6B, immunoreactive hEPO was readily detectable in the blood ofimplanted animals (the sensitivity of the hEPO ELISA has been determinedto be 2 mU/ml by the kit's manufacturer (R and D Systems) and endogenousmouse EPO shows no cross-reactivity with the antibodies used in theELISA kit). hEPO levels in the implanted animals dropped gradually, from29 to 9 mU/ml, from days 7 to 63 post-implantation.

[0118] This Example clearly demonstrates that normal skin fibroblaststhat have been genetically engineered to express and secrete hEPOcan: 1) survive in vivo to deliver hEPO to an animals systemiccirculation for up to 2 months, and 2) the hEPO produced is biologicallyfunctional, serving to prevent the drop in hematocrit observed in thefrequently bled control animals, and resulting in a net increase in HCTeven when animals were challenged with a bleeding schedule that producesan anemic response in control animals.

Example 11 Expression of GLP-1(7-37) From Secondary Human SkinFibroblasts Strains After Transfection With a GLP-1(7-37) ExpressionPlasmid

[0119] The portion of GLP-1 from amino acid residues 7 to 37[GLP-1(7-37); encoded by base pairs 7214 to 7306 in Genbank sequenceHUMGLUCG2] has been demonstrated to have insulinotropin activity invivo. Plasmid pXGLP1 is constructed such that the GLP-1(7-37) moiety isfused at its N-terminus to a 26-amino acid signal peptide derived fromhuman growth hormone for efficient transport through the endoplasmicreticulum. The fusion protein is cleaved immediately C-terminal toresidue 26 prior to secretion, such that the secreted product consistssolely of residues 7-37 of GLP-1. Expression of the signal peptide:GLP-1(1-37) fusion protein is controlled by the mouse metallothioneinpromoter.

[0120] Plasmid pXGLP1 is constructed as follows: Plasmid PXGH5 [Selden,R. F. et al., Mol. Cell. Biol. 6:3173-3179 (1986)] is digested with SmaIand ligated to a double-stranded oligonucleotide containing a BgIII site(BgIII linkers; New England Biolabs). The ligated product is digestedwith BgIII and EcoRI and the 0.32 kb fragment corresponding to the3′-untranslated region of the human growth hormone gene is isolated(with a BgIII linker attached to the SmaI site lying at position 6698 inGenbank entry HUMGHCSA). The hGH fragment can also be isolated by knownmethods from human genomic DNA using PCR primers designed to amplify thesequence between positions 6698 to 7321 in Genbank entry HUMGHCSA. A1.45 EcoRI-BgIII fragment containing the mouse metallothionein (mMT)promoter [Hamer, D. H. and Walling, M., J. Mol. Appl. Gen., 1:273-288(1982)] is next isolated. The mouse metallothionein promoter may beisolated by known methods from mouse genomic DNA using PCR primersdesigned from analysis of mMT sequences available from Genbank (i.e.Genbank entries MUSMTI, MUSMTIP, and MUSMTIPRM). Plasmid pUC19 (ATCC#37254) is digested with EcoRI and treated with bacterial alkalinephosphatase. The treated plasmid is ligated with the hGH and mMTfragments described above. The resulting plasmid has a single copy ofeach the mouse metallothionein promoter and the 3′untranslated region ofhGH joined at a BgIII site. This plasmid, designated pX1 is digestedwith BgIII and the full-length linear product is purified by gelelectrophoresis.

[0121] Oligonucleotides 11.1 and 11.2 are used to amplify a DNA sequenceencoding amino acids 7-37 of GLP-1 from human genomic DNA by PCR. Theamplified product (104 bp) is purified and mixed with pXGH5 andoligonucleotides 11.2, 11.3, 11.4, and 11.5 and subject to PCR.Oligonucleotides 11.3 and 11.4 are complementary and correspond to thedesired junction between the hGH signal peptide and GLP-1 amino acidresidue 7. The 500 base pair amplification product contains5′-untranslated, exon 1, intron 1, and part of exon 2 sequences from hGH(nucleotides 5168 to 5562 in Genbank entry HUMGHCSA) fused to a sequenceencoding GLP-1 residues 7-37 followed by a stop codon. The fragment, bydesign, is flanked on both ends by BamHI sites.

[0122] The fragment is cleaved with BamHI and ligated to the BgIIIdigest of pX1 described above. Ligation products are analysed toidentify those with one copy of the hGH-GLP-1(7-37) fusion productinserted at the BgIII site separating the mMT promoter and the3′-untranslated hGH sequence in pX1, such that GLP-1 residue 37 isdistal to the mMT promoter.

[0123] Oligonucleotides for Amplification of hGH-GLP-1(7-37) Fusion Gene11.1 5′CATGCTGAAG GGACCTTTAC CAGT (Seq. ID No.3) 11.2 5′TTGGATCCTTATCCTCGGCC TTTCACCAGC CA (Seq. ID No.4)           BamHI 11.35′GGCTTCAAGA GGGCAGTGCC CATGCTGAAG GGACCTTTAC CAGT (Seq. ID No.5) 11.45′ACTGGTAAAG GTCCCTTCAG CATGGGCACT GCCCTCTTGA AGCC (Seq. ID No.6) 11.55′AAGGATCCCA AGGCCCAACT CCCCGAAC (Seq. ID No.7)           BamHI 11.65′TTGGATCCTT ATCGGCC TTTCACCAGC CA (Seq. ID No.8)           BamHI

[0124] Alternatively, the small sizes of the signal peptide and GLP-1moieties needed allow complete fusion genes to be preparedsynthetically. DNA encoding the signal peptides of the LDL receptor(amino acid residues 1-21), preproglucagon (amino acid residues 1-20),or human growth hormone (amino acid residues 1-26) may be synthesized byknown methods and ligated in vitro to similarly synthesized DNA encodingamino acids 7-37 or 7-36 of GLP-1 (followed immediately by a stopcodon). The sequences necessary to design and synthesize these moleculesare readily available in Genbank entries HUMLDLR01 (human LDL receptor),HUMGLUCG2 (human GLP-1 and glucagon sequences), and HUMGHCSA (humangrowth hormone). The ligated product may be inserted into a suitablemammalian expression vector for use in human fibroblasts. Plasmid pMSG(Pharmacia LKB Biotechnology, Piscataway, N.J.) is suitable for thispurpose, having 5′ and 3′untranslated sequences, a splice site, a polyAaddition site, and an enhancer and promoter for use in human skinfibroblasts. Alternatively, the ligated product may be synthesized withan appropriate 5′-untranslated sequence and inserted into plasmid pX1described above.

[0125] A second insulinotropic GLP-1 derivative, GLP-1(7-36), can beexpressed by substituting oligonucleotide 11.6 for oligonucleotide 11.2described above. All subsequent cloning operations described above forconstruction of pXGLP1 are followed, such that the final product islacking the C-terminal glycine residue characteristic of GLP-1(7-37).Alternatively, this terminal glycine residue may be removed in vivo bythe activity of a peptidyl-glycine alpha-amidating enzyme to produce theinsulinotropin GLP-1(7-36) amide.

[0126] Plasmid pXGLP1 is co-transfected into primary human skinfibroblasts with plasmid pcDNEO exactly as described for pXEPO1 andpcDNEO in Example 5. Clones are selected in G418 containing medium,transferred to 96-well plates, and assayed for GLP-1(7-37) activity orimmunoreactivity in cell supernatants. GLP-1(7-37) activity isdetermined by incubation of cell supernatants with rat insulinoma RINm5Fcells and measuring the ability of the supernatants to induce insulinsecretion from these cells using a commercially available insulinradioimmunoassay (Coat-a-Count Insulin, DPC, Los Angeles, Calif.).GLP-1(7-37) antigen is determined using a commercially availableantisera against GLP-1 (Peninsula Laboratories, Belmont, Calif.).GLP-1(7-37) positive clones are expanded for implantation into nude miceas described in Example 10 and blood samples are taken to monitor serumhuman GLP-1(7-37) levels.

[0127] In vivo activity is monitored in fasting animals by determiningthe insulinogenic index after intraperitoneal injection of glucose (1 mgglucose per gram of body weight). Typically, implanted and non-implantedgroups of 32 mice are fasted overnight, and 28 are injected withglucose. After injection, the 28 mice are arbitrarily assigned to sevengroups of four, and blood sampling (for serum glucose and insulin) isperformed on each group at 5, 10, 20, 30, 45, 60, or 90 minutespost-injection, with the non-glucose injected group serving as a fastingcontrol. Increases in the postinjection insulinogenic index (the rationof insulin to glucose in the blood) in animals receiving GLP-1(7-37)expressing cells over non-implanted animals provides in vivo support forthe insulinotropic activity of the expressed peptide.

[0128] Equivalents

[0129] Those skilled in the art will recognize, or be able to ascertainusing not more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0130] Statement Regarding Correspondence of Sequence Information (PaperCopy and Disk)

[0131] A sequence listing in computer readable form and in paper formare being filed with this application. As required by 37 C.F.R. 1.821(f), Applicants' Attorney hereby states that the content of the SequenceListing in paper from and of the computer readable form of the “SequenceListing” are the same.

1 8 1 31 DNA Homo sapiens 1 cccatattac gtttgctcag cttggtgctt g 31 2 30DNA Homo sapiens 2 cccatattac tcaagttggc cctgtgacat 30 3 34 DNA Homosapiens 3 cccatattac catgctgaag ggacctttac cagt 34 4 42 DNA Homo sapiens4 cccatattac ttggatcctt atcctcggcc tttcaccagc ca 42 5 54 DNA Homosapiens 5 cccatattac ggcttcaaga gggcagtgcc catgctgaag ggacctttac cagt 546 54 DNA Homo sapiens 6 cccatattac actggtaaag gtcccttcag catgggcactgccctcttga agcc 54 7 38 DNA Homo sapiens 7 cccatattac aaggatcccaaggcccaact ccccgaac 38 8 39 DNA Homo sapiens 8 cccatattac ttggatccttatcggccttt caccagcca 39

1. A transfected primary or secondary cell of vertebrate origin havingstably integrated into its genome: a) exogenous DNA which encodeserythropoietin and b) DNA sequences, sufficient for expression of theexogenous DNA in the transfected primary or secondary cell, the primaryor secondary cell capable of expressing erythropoietin.
 2. Thetransfected primary or secondary cell of vertebrate origin of claim 1selected from the group consisting of: transfected fibroblasts,transfected keratinocytes, transfected epithelial cells, transfectedendothelial cells, transfected glial cells, transfected neural cells,transfected formed elements of the blood, transfected muscle cells,transfected hepatocytes, and transfected precursors thereof.
 3. Thetransfected primary or secondary cell of claim 2 which is of mammalianorigin.
 4. The transfected primary or secondary cell of claim 3 selectedfrom the group consisting of: transfected primary human cells,transfected secondary human cells, transfected primary rabbit cells andtransfected secondary rabbit cells.
 5. The transfected primary orsecondary cell of vertebrate origin of claim 1 which additionallyincludes DNA encoding a selectable marker.
 6. The transfected primary orsecondary cell of claim 5 selected from the group consisting of:transfected fibroblasts, transfected keratinocytes, transfectedepithelial cells, transfected endothelial cells, transfected glialcells, transfected neural cells, transfected formed elements of theblood, transfected muscle cells, transfected hepatocytes, andtransfected precursors thereof.
 7. The transfected primary or secondarycell of claim 6 which is of mammalian origin.
 8. The transfected primaryor secondary cell of claim 7 selected from the group consisting of:transfected primary human cells, transfected secondary human cells,transfected primary rabbit cells, and transfected secondary rabbitcells.
 9. The transfected primary or secondary cell of vertebrate originof claim 1 selected from the group consisting of: a) transfected primaryor secondary cells which, in their untransfected state, do not make orcontain erythropoietin; b) transfected primary or secondary cells which,in their untransfected state, make or contain erythropoietin inabnormally low amounts or in defective form; and c) transfected primaryor secondary cells which, in their untransfected form, make or containerythropoietin in physiologically normal amounts.
 10. A primary orsecondary cell of vertebrate origin transfected with: a) exogenous DNAwhich encodes erythropoietin; and b) DNA sequences, sufficient forexpression of the exogenous DNA in the primary or secondary cell, thesequences of (a) and (b) present in the cell in an episome.
 11. Theprimary or secondary cell of vertebrate origin of claim 10 selected fromthe group consisting of: fibroblasts, keratinocytes, epithelial cells,endothelial cells, glial cells, neural cells, formed elements of theblood, muscle cells, hepatocytes, and precursors thereof.
 12. Theprimary or secondary cell of claim 11 which is of mammalian origin. 13.The primary or secondary cell of claim 12 selected from the groupconsisting of: primary human cells, secondary human cells, primaryrabbit cells, and secondary rabbit cells.
 14. A clonal cell strain oftransfected secondary cells of vertebrate origin which express exogenousDNA encoding erythropoietin incorporated therein.
 15. The clonal cellstrain of claim 14 wherein the exogenous DNA is stably incorporated intogenomic DNA of the transfected secondary cells.
 16. The clonal cellstrain of claim 15 wherein the transfected secondary cells are selectedfrom the group consisting of: transfected secondary fibroblasts,transfected secondary keratinocytes, transfected epithelial cells,transfected endothelial cells, transfected glial cells, transfectedneural cells, transfected formed elements of the blood, transfectedmuscle cells, transfected hepatocytes, and transfected precursorsthereof.
 17. The clonal cell strain of transfected secondary cells ofclaim 16 wherein the transfected secondary cells are of mammalianorigin.
 18. The clonal strain of transfected secondary cells of claim 17wherein the transfected secondary cells of mammalian origin are selectedfrom the group consisting of: transfected secondary human cells andtransfected secondary rabbit cells.
 19. The clonal cell strain of claim14 wherein the exogenous DNA is present in the transfected secondarycells in an episome.
 20. A heterogenous cell strain of transfectedsecondary cells of vertebrate origin having stably incorporated intotheir genomes: a) exogenous DNA encoding erythropoietin and b) DNAsequences sufficient for expression of the exogenous DNA in thetransfected primary or secondary cell. the heterogenous cell straincapable of expressing erythropoietin.
 21. The heterogenous cell strainof claim 20, wherein the transfected primary or secondary cells areselected from the group consisting of: transfected fibroblasts,transfected keratinocytes, transfected epithelial cells, transfectedendothelial cells, transfected glial cells, transfected neural cells,transfected formed elements of the blood, transfected muscle cells,transfected hepatocytes, and transfected precursors thereof.
 22. Theheterogenous cell strain of claim 21 which is of mammalian origin.
 23. Aheterogenous cell strain of claim 22 selected from the group consistingof: transfected primary human cells, transfected secondary human cells,transfected primary rabbit cells, and transfected secondary rabbitcells.
 24. A mixture of cells consisting essentially of transfectedprimary or secondary cells of claim 1 and untransfected primary orsecondary cells.
 25. A mixture of cells consisting essentially oftransfected primary or secondary cells of claim 3 and untransfectedprimary or secondary cells.
 26. A method of producing a clonal cellstrain of transfected secondary cells of vertebrate origin which expressexogenous DNA encoding erythropoietin incorporated therein, comprisingthe steps of: a) producing a mixture of cells of vertebrate origincontaining primary cells; b) transfecting primary cells produced in (a)with a DNA construct comprising exogenous DNA encoding erythropoietinand additional DNA sequences sufficient for expression of the exogenousDNA in the primary cells, thereby producing transfected primary cellswhich express the exogenous DNA encoding erythropoietin; c) culturing atransfected primary cell which expresses the exogenous DNA encodingerythropoietin produced in (b), under conditions appropriate forpropagating the transfected primary cell which expresses the exogenousDNA encoding erythropoietin, thereby producing a clonal cell strain oftransfected secondary cells from the transfected primary cell.
 27. Themethod of claim 26 wherein the primary cells are selected from the groupconsisting of: fibroblasts, keratinocytes, epithelial cells, endothelialcells, glial cells, neural cells, formed elements of the blood, musclecells, hepatocytes, and precursors thereof.
 28. The method of claim 27wherein the transfected primary cell is of mammalian origin.
 29. Themethod of claim 28 wherein the primary cell is selected from the groupconsisting of: primary human cells, and primary rabbit cells.
 30. Themethod of claim 26 wherein in step (b) the primary cell of vertebrateorigin is additionally transfected with DNA encoding a selectablemarker.
 31. The method of claim 30 wherein the primary cell is selectedfrom the group consisting of: fibroblasts, keratinocytes, epithelialcells, endothelial cells, glial cells, neural cells, formed elements ofthe blood, transfected muscle cells, hepatocytes, and precursorsthereof.
 32. The method of claim 31 wherein the primary cell is ofmammalian origin.
 33. The method of claim 32 wherein the primary cell isselected from the group consisting of: primary human cells and primaryrabbit cells.
 34. A method of producing a clonal cell strain oftransfected secondary cells of vertebrate origin which express exogenousDNA encoding erythropoietin incorporated therein, comprising the stepsof: a) providing a mixture of cells of vertebrate origin containingprimary cells; b) producing a population of secondary cells from theprimary cells provided in (a); c) transfecting secondary cells producedin (b) with a DNA construct comprising exogenous DNA encodingerythropoietin and additional DNA sequences sufficient for expression ofthe exogenous DNA in the secondary cells, thereby producing transfectedsecondary cells which express the exogenous DNA encoding erythropoietin;and d) culturing a transfected secondary cell which expresses the DNAencoding erythropoietin, produced in (c), under conditions appropriatefor propagating the transfected secondary cell which expresses theexogenous DNA encoding erythropoietin, thereby producing a clonal cellstrain of transfected secondary cells from the transfected secondarycell of (d).
 35. The method of claim 34 wherein the primary cells areselected from the group consisting of: fibroblasts, keratinocytes,epithelial cells, endothelial cells, glial cells, neural cells, formedelements of the blood, muscle cells, hepatocytes, and precursorsthereof.
 36. The method of claim 35 wherein the transfected primary cellis of mammalian origin.
 37. The method of claim 36 wherein the primarycell is selected from the group consisting of: primary human cells andprimary rabbit cells.
 38. The method of claim 34 wherein in step (c) thesecondary cells of vertebrate origin are additionally transfected withDNA encoding a selectable marker.
 39. The method of claim 38 wherein theprimary cell is selected from the group consisting of: fibroblasts,keratinocytes, epithelial cells, endothelial cells, glial cells, neuralcells, formed elements of the blood, transfected muscle cells,hepatocytes, and precursors thereof.
 40. The method of claim 39 whereinthe primary cell is of mammalian origin.
 41. The method of claim 40wherein the primary cell is selected from the group consisting of:primary human cells and primary rabbit cells.
 42. The method of claim 34wherein, in step (c), secondary cells are transfected with the DNAconstruct comprising exogenous DNA encoding erythropoietin by combiningthe primary cells and the DNA construct comprising exogenous DNAencoding erythropoietin and subjecting the resulting combination toelectroporation under conditions which result in production of at leastone primary cell having exogenous DNA stably integrated into genomicDNA.
 43. The method of claim 42 wherein electroporation is carried outat an electroporation voltage of between 250 and 300 volts and acapacitance setting of approximately 960 μFarads.
 44. The method ofclaim 34 wherein in step (c) secondary cells are transfected with theDNA construct comprising exogenous DNA by microinjecting the DNAconstruct comprising exogenous DNA into the secondary cells.
 45. Themethod of claim 34 wherein in step (c), secondary cells are transfectedwith the DNA construct comprising exogenous DNA by calcium phosphateprecipitation, modified calcium phosphate precipitation, liposome fusionmethodologies, receptor mediated transfer, micro-projectile bombardment,and polybrene precipitation.
 46. The method of claim 34 wherein in step(c), the exogenous DNA is introduced into genomic DNA by homologousrecombination between DNA sequences present in the exogenous DNAconstruct and genomic DNA.
 47. The method of claim 34 additionallycomprising transfecting in step (c), second cells produced in step (a)with a DNA construct comprising DNA encoding a selectable marker.
 48. Amethod of producing a heterogenous cell strain of transfected secondarycells of vertebrate origin which express exogenous DNA encodingerythropoietin stably incorporated into the genome of said secondarycells, comprising the steps of: a) producing a mixture of cells ofvertebrate origin containing primary cells; b) transfecting primarycells produced in (a) with exogenous DNA encoding erythropoietin andoperatively linked to DNA sequences of non-retroviral origin sufficientfor expression of the exogenous DNA in transfected secondary cells,thereby producing a mixture of primary cells which includes transfectedprimary cells which express the exogenous DNA encoding erythropoietin;c) culturing the product of (b) under conditions appropriate forpropagation of transfected primary cells which express the exogenous DNAencoding erythropoietin, thereby producing a heterogenous cell strain oftransfected secondary cells of vertebrate origin which express theexogenous DNA encoding erythropoietin.
 49. The method of claim 48wherein the vertebrate is a mammal and and the primary cells areselected from the group consisting of: fibroblasts, keratinocytes,epithelial cells, endothelial cells, glial cells, neural cells, formedelements of the blood, muscle cells, hepatocytes, and precursorsthereof.
 50. The method of claim 48 wherein, in step (b), primary cellsare transfected with the DNA construct comprising exogenous DNA encodinga therapeutic product by combining the primary cells and the DNAconstruct comprising exogenous DNA encoding a therapeutic product andsubjecting the resulting combination to electroporation under conditionswhich result in production of at least one secondary cell havingexogenous DNA stably integrated into genomic DNA.
 51. The method ofclaim 50 wherein electroporation is carried out at an electroporationvoltage of between 250 and 300 volts and a capacitance setting ofapproximately 960 μFarads.
 52. The method of claim 48 wherein in step(b) primary cells are transfected with the DNA construct comprisingexogenous DNA by microinjecting the DNA construct comprising exogenousDNA into the primary cells.
 53. The method of claim 48 wherein in step(b), secondary cells are transfected with the DNA construct comprisingexogenous DNA by a method selected from the group consisting of: calciumphosphate precipitation, modified calcium phosphate precipitation,liposome fusion methodologies, receptor mediated transfer,micro-projectile bombardment, and polybrene precipitation.
 54. Themethod of claim 48 wherein in step (b) transfected primary cells areproduced by introducing into primary cells produced in (a) a constructwhich undergoes homologous recombination with genomic DNA of the primarycells, thereby resulting in introduction of the construct into thegenomic DNA.
 55. The method of claim 48 additionally comprisingtransfecting in step (b), primary cells produced in step (a) with a DNAconstruct comprising DNA encoding a selectable marker.
 56. A method ofproducing a heterogenous cell strain of transfected secondary cells ofvertebrate origin which express exogenous DNA encoding erythropoietinstably incorporated into the genome of said secondary cells, comprisingthe steps of a) providing a mixture of cells of vertebrate origincontaining primary cells; b) producing a population of secondary cellsfrom the primary cells provided in (a); c) transfecting secondary cellsproduced in (b) with exogenous DNA encoding erythropoietin andoperatively linked to DNA sequences of non-retroviral origin sufficientfor expression of the exogenous DNA in transfected secondary cells,thereby producing a mixture of secondary cells which includestransfected secondary cells which express the exogenous DNA encodingerythropoietin; d) culturing the product of (c) under conditionsappropriate for propagation of transfected secondary cells which expressthe exogenous DNA encoding a therapeutic product, thereby producing aheterogenous cell strain of transfected secondary cells of vertebrateorigin which express the exogenous DNA encoding erythropoietin.
 57. Themethod of claim 56 wherein the vertebrate is a mammal and and theprimary cells are selected from the group consisting of: fibroblasts,keratinocytes, epithelial cells, endothelial cells, glial cells, neuralcells, formed elements of the blood, muscle cells, hepatocytes andprecursors thereof.
 58. The method of claim 56 wherein, in step (c),secondary cells are transfected with the DNA construct comprisingexogenous DNA encoding a therapeutic product by combining the primarycells and the DNA construct comprising exogenous DNA encoding atherapeutic product and subjecting the resulting combination toelectroporation under conditions which result in production of at leastone secondary cell having exogenous DNA stably integrated into genomicDNA.
 59. The method of claim 58 wherein electroporation is carried outat an electroporation voltage of between 250 and 300 volts and acapacitance setting of approximately 960 μFarads.
 60. The method ofclaim 56 wherein in step (c) secondary cells are transfected with theDNA construct comprising exogenous DNA by microinjecting the DNAconstruct comprising exogenous DNA into the secondary cells.
 61. Themethod of claim 56 wherein in step (c), secondary cells are transfectedwith the DNA construct comprising exogenous DNA by a method selectedfrom the group consisting of: calcium phosphate precipitation, modifiedcalcium phosphate precipitation, liposome fusion methodologies, receptormediated transfer, micro-projectile bombardment, and polybreneprecipitation.
 62. The method of claim 58, wherein in step (c)transfected secondary cells are produced by introducing into secondarycells produced in (b) a construct which undergoes homologousrecombination with genomic DNA of the secondary cells, thereby resultingin introduction of the construct into the genomic DNA.
 63. The method ofclaim 60, wherein in step (c) transfected secondary cells are producedby introducing into secondary cells produced in (b) a construct whichundergoes homologous recombination with genomic DNA of the secondarycells, thereby resulting in introduction of the construct into thegenomic DNA.
 64. The method of claim 56 additionally comprisingtransfecting in step (c), secondary cells produced in step (b) with aDNA construct comprising DNA encoding a selectable marker.
 65. A methodof producing a clonal cell strain of secondary fibroblasts of mammalianorigin which express exogenous DNA encoding erythropoietin uponintroduction into a mammal, comprising the steps of: a) providingprimary fibroblasts of mammalian origin; b) producing a population ofsecondary fibroblasts from the primary fibroblasts provided in (a); c)combining the secondary fibroblasts of mammalian origin with a DNAconstruct comprising: i) exogenous DNA encoding erythropoietin to beexpressed in the fibroblasts; and ii) additional DNA sequences ofnon-retroviral origin sufficient for expression of the exogenous DNA inthe fibroblasts; d) subjecting the combination produced in (c) toelectroporation under conditions which result in transfection of thevector into the secondary fibroblasts of mammalian origin, therebyproducing a mixture of transfected secondary fibroblasts of mammalianorigin and non-transfected secondary fibroblasts of mammalian origin; e)isolating a transfected secondary fibroblast of mammalian originproduced in (d); and f) culturing the transfected secondary fibroblastof mammalian origin isolated in (e) under conditions appropriate forproduction of a clonal population consisting essentially of transfectedsecondary fibroblasts of mammalian origin which express the exogenousDNA encoding erythropoietin.
 66. The method of claim 65 wherein in step(d) electroporation is carried out at an electroporation voltage ofbetween 250 and 300 volts and a capacitance setting of approximately 960μFarads.
 67. The method of claim 65 further comprising maintaining theproduct of (f) for sufficient time and under appropriate conditions forat least 20 doublings of the transfected secondary cells which expressthe exogenous DNA to occur.
 68. A method of providing erythropoietin inan effective amount to a mammal, comprising the steps of: a) obtaining asource of primary cells from the mammal; b) transfecting primary cellsobtained in (a) with a DNA construct comprising exogenous DNA encodingerythropoietin and additional DNA sequences sufficient for expression ofthe exogenous DNA in the primary cells, thereby producing transfectedprimary cells which express the exogenous DNA encoding the therapeuticproduct; c) culturing a transfected primary cell which expresses theexogenous DNA encoding erythropoietin produced in (b), under conditionsappropriate for propagating the transfected primary cell which expressesthe exogenous DNA encoding erythropoietin, thereby producing a clonalcell strain of transfected secondary cells from the transfected primarycell; d) culturing the clonal cell strain of transfected secondary cellsproduced in (c) under conditions appropriate for and sufficient time forthe clonal cell strain of transfected secondary cells to undergo asufficient number of doublings to provide a sufficient number oftransfected secondary cells to produce an effective amount oferythropoietin; and e) introducing transfected secondary cells producedin (d) into the mammal in sufficient number to produce an effectiveamount of erythropoietin in the mammal.
 69. The method of providingerythropoietin in an effective amount to a mammal of claim 68 whereinthe primary cells are selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, formed elements of the blood, hepatocytes, andprecursors thereof.
 70. A method of providing erythropoietin in aneffective amount to a mammal, comprising the steps of: a) obtaining asource of primary cells from the mammal; b) producing a population ofsecondary cells from the primary cells provided in (a); c) transfectingsecondary cells produced in (b) with a DNA construct comprisingexogenous DNA encoding erythropoietin and additional DNA sequencessufficient for expression of the exogenous DNA in the primary cells,thereby producing transfected secondary cells which express theexogenous DNA encoding erythropoietin; d) culturing a transfectedsecondary cell which expresses the exogenous DNA encoding erythropoietinproduced in (c), under conditions appropriate for propagating thetransfected secondary cell which expresses the exogenous DNA encodingerythropoietin, thereby producing a clonal cell strain of transfectedsecondary cells from the transfected secondary cell; e) culturing theclonal cell strain of transfected secondary cells produced in (d) underconditions appropriate for and sufficient time for the clonal cellstrain of transfected secondary cells to undergo a sufficient number ofdoublings to provide a sufficient number of transfected secondary cellsto produce an effective amount of erythropoietin; and f) introducingtransfected secondary cells produced in (e) into the mammal insufficient number to produce an effective amount of erythropoietin. 71.The method of providing a therapeutic product in an effective amount toa mammal of claim 70 wherein the primary cells are selected from thegroup consisting of: fibroblasts, keratinocytes, epithelial cells,endothelial cells, glial cells, neural cells, formed elements of theblood, hepatocytes and precursors thereof.
 72. A method of producingerythropoietin in an effective amount to a mammal, comprising the stepsof: a) obtaining a source of primary cells from the mammal; b)transfecting primary cells obtained in (a) with exogenous DNA encodingerythropoietin and operatively linked to DNA sequences of non-retroviralorigin sufficient for expression of the exogenous DNA in transfectedsecondary cells, thereby producing a mixture of primary cells whichincludes transfected primary cells which express the exogenous DNAencoding erythropoietin; c) culturing the product of (b) underconditions appropriate for propagation of transfected primary cellswhich express the exogenous DNA encoding erythropoietin, therebyproducing a heterogenous cell strain of transfected secondary cells ofvertebrate origin which express the exogenous DNA encodingerythropoietin; and d) introducing transfected secondary cells producedin (c) into the mammal in sufficient number to produce an effectiveamount of erythropoietin in the mammal.
 73. The method of claim 72,wherein the primary cells are selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, formed elements of the blood, hepatocytes, andprecursors thereof.
 74. A method of providing erythropoietin in aneffective amount to a mammal, comprising the steps of: a) obtaining asource of primary cells from the mammal; b) producing a population ofsecondary cells from the primary cells provided in (a); c) transfectingsecondary cells produced in (b) with exogenous DNA encodingerythropoietin and operatively linked to DNA sequences of non-retroviralorigin sufficient for expression of the exogenous DNA in transfectedsecondary cells, thereby producing a mixture of secondary cells whichincludes transfected secondary cells which express the exogenous DNAencoding erythropoietin; d) culturing the product of (c) underconditions appropriate for propagation of transfected secondary cellswhich express the exogenous DNA encoding erythropoietin, therebyproducing a heterogenous cell strain of transfected secondary cells ofvertebrate origin which express the exogenous DNA encodingerythropoietin; and e) introducing transfected secondary cells producedin (c) into the mammal in sufficient number to produce an effectiveamount of erythropoietin in the mammal.
 75. The method of claim 74wherein the primary cells are selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, formed elements of the blood, hepatocytes, andprecursors thereof.
 76. A method of providing erythropoietin to a mammalat biologically significant levels, comprising administering to themammal transfected primary or secondary cells of mammalian origin whichexpress erythropoietin in sufficient quantity to produce physiologicallyrelevant levels in the mammal.
 77. The method of claim 76 wherein thetransfected primary or secondary cells are selected from the groupconsisting of primary human cells, primary rabbit cells.
 78. Atransfected primary or secondary cell of vertebrate origin having stablyintegrated into its genome: a) exogenous DNA which encodes aglucagon-like peptide 1 related peptide with insulinotropin activity,and b) DNA sequences, sufficient for expression of the exogenous DNA inthe transfected primary or secondary cell, the primary or secondary cellcapable of expressing the glucagon-like peptide 1 related peptide. 79.The transfected primary or secondary cell of vertebrate origin of claim78 selected from the group consisting of: transfected fibroblasts,transfected keratinocytes, transfected epithelial cells, transfectedendothelial cells, transfected glial cells, transfected neural cells,transfected formed elements of the blood, transfected muscle cells,transfected hepatocytes, and transfected precursors thereof.
 80. Thetransfected primary or secondary cell of claim 79 which is of mammalianorigin.
 81. The transfected primary or secondary cell of claim 80selected from the group consisting of: transfected primary human cells,transfected secondary human cells, transfected primary rabbit cells andtransfected secondary rabbit cells.
 82. The transfected primary orsecondary cell of vertebrate origin of claim 78 which additionallyincludes DNA encoding a selectable marker.
 83. The transfected primaryor secondary cell of claim 82 selected from the group consisting of:transfected fibroblasts, transfected keratinocytes, transfectedepithelial cells, transfected endothelial cells, transfected glialcells, transfected neural cells, transfected formed elements of theblood, transfected muscle cells, transfected hepatocytes and transfectedprecursors thereof.
 84. The transfected primary or secondary cell ofclaim 83 which is of mammalian origin.
 85. The transfected primary orsecondary cell of vertebrate origin of claim 78 selected from the groupconsisting of: a) transfected primary or secondary cells which, in theiruntransfected state, do not make or contain a glucagon-like peptide 1related peptide; b) transfected primary or secondary cells which, intheir untransfected state, make or contain a glucagon-like peptide 1related peptide in abnormally low amounts or in defective form; and c)transfected primary or secondary cells which, in their untransfectedform, make or contain a glucagon-like peptide 1 related peptide inphysiologically normal amounts.
 86. A primary or secondary cell ofvertebrate origin transfected with: a) exogenous DNA which encodes aglucagon-like peptide 1 related peptide with insulinotropin activity;and b) DNA sequences, sufficient for expression of the exogenous DNA inthe primary or secondary cell, the sequences of (a) and (b) present inthe cell in an episome.
 87. A clonal cell strain of transfectedsecondary cells of vertebrate origin which express exogenous DNAencoding a glucagon-like peptide 1 related peptide incorporated therein.88. The clonal cell strain of claim 87 wherein the exogenous DNA isstably incorporated into genomic DNA of the transfected secondary cells.89. A heterogenous cell strain of transfected secondary cells ofvertebrate origin having stably incorporated into their genomes: a)exogenous DNA encoding a glucagon-like peptide 1 related peptide withinsulinotropin activity and b) DNA sequences sufficient for expressionof the exogenous DNA in the transfected primary or secondary cell, theheterogenous cell strain capable of expressing a glucagon-like peptide 1related peptide.
 90. A mixture of cells consisting essentially oftransfected primary or secondary cells of claim 78 and untransfectedprimary or secondary cells.
 91. A method of producing a clonal cellstrain of transfected secondary cells of vertebrate origin which expressexogenous DNA encoding a glucagon-like peptide 1 related peptideincorporated therein, comprising the steps of: a) producing a mixture ofcells of vertebrate origin containing primary cells; b) transfectingprimary cells produced in (a) with a DNA construct comprising exogenousDNA encoding a glucagon-like peptide 1 related peptide and additionalDNA sequences sufficient for expression of the exogenous DNA in theprimary cells, thereby producing transfected primary cells which expressthe exogenous DNA encoding a glucagon-like peptide 1 related peptide;and c) culturing a transfected primary cell which expresses theexogenous DNA encoding a glucagon-like peptide 1 related peptideproduced in (b), under conditions appropriate for propagating thetransfected primary cell which expresses the exogenous DNA encoding aglucagon-like peptide 1 related peptide, thereby producing a clonal cellstrain of transfected secondary cells from the transfected primary cell.92. A method of producing a clonal cell strain of transfected secondarycells of vertebrate origin which express exogenous DNA encoding aglucagon-like peptide 1 related peptide incorporated therein, comprisingthe steps of: a) providing a mixture of cells of vertebrate origincontaining primary cells; b) producing a population of secondary cellsfrom the primary cells provided in (a); c) transfecting secondary cellsproduced in (b) with a DNA construct comprising exogenous DNA encoding aglucagon-like peptide 1 related peptide and additional DNA sequencessufficient for expression of the exogenous DNA in the secondary cells,thereby producing transfected secondary cells which express theexogenous DNA encoding a glucagon-like peptide 1 related peptide; and d)culturing a transfected secondary cell which expresses the DNA encodinga glucagon-like peptide 1 related peptide produced in (c), underconditions appropriate for propagating the transfected secondary cellwhich expresses the exogenous DNA encoding a glucagon-like peptide 1related peptide, thereby producing a clonal cell strain of transfectedsecondary cells from the transfected secondary cell of (d).
 93. A methodof producing a heterogenous cell strain of transfected secondary cellsof vertebrate origin which express exogenous DNA encoding aglucagon-like peptide 1 related peptide stably incorporated into thegenome of said secondary cells, comprising the steps of: a) producing amixture of cells of vertebrate origin containing primary cells; b)transfecting primary cells produced in (a) with exogenous DNA encoding aglucagon-like peptide 1 related peptide and operatively linked to DNAsequences of non-retroviral origin sufficient for expression of theexogenous DNA in transfected secondary cells, thereby producing amixture of primary cells which includes transfected primary cells whichexpress the exogenous DNA encoding a glucagon-like peptide 1 relatedpeptide; c) culturing the product of (b) under conditions appropriatefor propagation of transfected primary cells which express the exogenousDNA encoding a glucagon-like peptide 1 related peptide, therebyproducing a heterogenous cell strain of transfected secondary cells ofvertebrate origin which express the exogenous DNA encoding aglucagon-like peptide 1 related peptide.
 94. A method of producing aclonal cell strain of secondary fibroblasts of mammalian origin whichexpress exogenous DNA encoding a glucagon-like peptide 1 related peptideupon incorporation into the genome of the secondary fibroblast,comprising the steps of: a) providing primary fibroblasts of mammalianorigin; b) producing a population of secondary fibroblasts from theprimary fibroblasts provided in (a); c) combining the secondaryfibroblasts of mammalian origin with a DNA construct comprising: i)exogenous DNA encoding a glucagon-like peptide 1 related peptide to beexpressed in the fibroblasts; and ii) additional DNA sequences ofnon-retroviral origin sufficient for expression of the exogenous DNA inthe fibroblasts; d) subjecting the combination produced in (c) toelectroporation under conditions which result in transfection of thevector into the secondary fibroblasts of mammalian origin, therebyproducing a mixture of transfected secondary fibroblasts of mammalianorigin and non-transfected secondary fibroblasts of mammalian origin; e)isolating a transfected secondary fibroblast of mammalian originproduced in (d); and f) culturing the transfected secondary fibroblastof mammalian origin isolated in (e) under conditions appropriate forproduction of a clonal population consisting essentially of transfectedsecondary fibroblasts of mammalian origin which express the exogenousDNA encoding a glucagon-like peptide 1 related peptide.
 95. A method ofclaim 94 wherein the glucagon-like peptide 1 related peptide is aglucan-like peptide 1 derivative selected from the group consisting ofGLP-1(7-37), GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and other truncatedcarboxy-terminal amidated derivatives and derivatives of GLP-1 whichhave amino acid substitutions, deletions, additions or other alterations(e.g., addition of a non-amino acid component) which result inbiological activity or stability in the blood which is substantially thesame as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood.
 96. A method of providing aglucagon-like peptide 1 related peptide in an effective amount to amammal, comprising the steps of: a) obtaining a source of primary cellsfrom the mammal; b) transfecting primary cells obtained in (a) with aDNA construct comprising exogenous DNA encoding a glucagon-like peptide1 related peptide and additional DNA sequences sufficient for expressionof the exogenous DNA in the primary cells, thereby producing transfectedprimary cells which express the exogenous DNA encoding a glucagon-likepeptide 1 related peptide; c) culturing a transfected primary cell whichexpresses the exogenous DNA encoding a glucagon-like peptide 1 relatedpeptide produced in (b), under conditions appropriate for propagatingthe transfected primary cell which expresses the exogenous DNA encodinga glucagon-like peptide 1 related peptide, thereby producing a clonalcell strain of transfected secondary cells from the transfected primarycell; d) culturing the clonal cell strain of transfected secondary cellsproduced in (c) under conditions appropriate for and sufficient time forthe clonal cell strain of transfected secondary cells to undergo asufficient number of doublings to provide a sufficient number oftransfected secondary cells to produce an effective amount of aglucagon-like peptide 1 related peptide; and e) introducing transfectedsecondary cells produced in (d) into the mammal in sufficient number toproduce an effective amount of a glucagon-like peptide 1 related peptidein the mammal.
 97. The method of providing a therapeutic product in aneffective amount to a mammal of claim 95 wherein the primary cells areselected from the group consisting of: fibroblasts, keratinocytes,epithelial cells, endothelial cells, glial cells, neural cells, formedelements of the blood, hepatocytes and precursors thereof.
 98. A methodof claim 97 wherein the glucagon-like peptide 1 related peptide is aglucan-like peptide 1 derivative selected from the group consisting ofGLP-1(7-37), GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and other truncatedcarboxy-terminal amidated derivatives and derivatives of GLP-1 whichhave amino acid substitutions, deletions, additions or other alterations(e.g., addition of a non-amino acid component) which result inbiological activity or stability in the blood which is substantially thesame as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood.
 99. A method of providing aglucagon-like peptide 1 related peptide in an effective amount to amammal, comprising the steps of: a) obtaining a source of primary cellsfrom the mammal; b) producing a population of secondary cells from theprimary cells provided in (a); c) transfecting secondary cells producedin (b) with a DNA construct comprising exogenous DNA encoding aglucagon-like peptide 1 related peptide and additional DNA sequencessufficient for expression of the exogenous DNA in the primary cells,thereby producing transfected secondary cells which express theexogenous DNA encoding glucagon-like peptide; d) culturing a transfectedsecondary cell which expresses the exogenous DNA encoding glucagon-likepeptide produced in (c), under conditions appropriate for propagatingthe transfected secondary cell which expresses the exogenous DNAencoding a glucagon-like peptide 1 related peptide, thereby producing aclonal cell strain of transfected secondary cells from the transfectedsecondary cell; e) culturing the clonal cell strain of transfectedsecondary cells produced in (c) under conditions appropriate for andsufficient time for the clonal cell strain of transfected secondarycells to undergo a sufficient number of doublings to provide asufficient number of transfected secondary cells to produce an effectiveamount of a glucagon-like peptide 1 related peptide; and f) introducingtransfected secondary cells produced in (e) into the mammal insufficient number to produce an effective amount of a glucagon-likepeptide 1 related peptide.
 100. A method of claim 99 wherein theglucagon-like peptide 1 related peptide is a glucan-like peptide 1derivative selected from the group consisting of GLP-1(7-37),GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and other truncatedcarboxy-terminal amidated derivatives and derivatives of GLP-1 whichhave amino acid substitutions, deletions, additions or other alterations(e.g., addition of a non-amino acid component) which result inbiological activity or stability in the blood which is substantially thesame as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood.
 101. A method of producing aglucagon-like peptide 1 related peptide in an effective amount to amammal, comprising the steps of: a) obtaining a source of primary cellsfrom the mammal; b) transfecting primary cells obtained in (a) withexogenous DNA encoding a glucagon-like peptide 1 related peptide andoperatively linked to DNA sequences of non-retroviral origin sufficientfor expression of the exogenous DNA in transfected secondary cells,thereby producing a mixture of primary cells which includes transfectedprimary cells which express the exogenous DNA encoding a glucagon-likepeptide 1 related peptide; c) culturing the product of (b) underconditions appropriate, for propagation of transfected primary cellswhich express the exogenous DNA encoding a glucagon-like peptide 1related peptide, thereby producing a heterogenous cell strain oftransfected secondary cells of vertebrate origin which express theexogenous DNA encoding a glucagon-like peptide 1 related peptide; and d)introducing transfected secondary cells produced in (c) into the mammalin sufficient number to produce an effective amount of a glucagon-likepeptide 1 related peptide in the mammal.
 102. A method of claim 101wherein the glucagon-like peptide 1 related peptide is a glucan-likepeptide 1 derivative selected from the group consisting of GLP-1(7-37),GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and other truncatedcarboxy-terminal amidated derivatives and derivatives of GLP-1 whichhave amino acid substitutions, deletions, additions or other alterations(e.g., addition of a non-amino acid component) which result inbiological activity or stability in the blood which is substantially thesame as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood.
 103. A method of providing aglucagon-like peptide 1 related peptide in an effective amount to amammal, comprising the steps of: a) obtaining a source of primary cellsfrom the mammal; b) producing a population of secondary cells from theprimary cells provided in (a); c) transfecting secondary cells producedin (b) with exogenous DNA encoding a glucagon-like peptide 1 relatedpeptide and operatively linked to DNA sequences of non-retroviral originsufficient for expression of the exogenous DNA in transfected secondarycells, thereby producing a mixture of secondary cells which includestransfected secondary cells which express the exogenous DNA encoding aglucagon-like peptide 1 related peptide; d) culturing the product of (c)under conditions appropriate for propagation of transfected secondarycells which express the exogenous DNA encoding a glucagon-like peptide 1related peptide, thereby producing a heterogenous cell strain oftransfected secondary cells of vertebrate origin which express theexogenous DNA encoding glucagon-like peptide; and e) introducingtransfected secondary cells produced in (c) into the mammal insufficient number to produce an effective amount of a glucagon-likepeptide 1 related peptide in the mammal.
 104. A method of claim 103wherein the glucagon-like peptide 1 related peptide is a glucan-likepeptide 1 derivative selected from the group consisting of GLP-1(7-37),GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and other truncatedcarboxy-terminal amidated derivatives and derivatives of GLP-1 whichhave amino acid substitutions, deletions, additions or other alterations(e.g., addition of a non-amino acid component) which result inbiological activity or stability in the blood which is substantially thesame as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood.
 105. A method of providingerythropoietin in an effective amount to a mammal, comprisingintroducing into the mammal a barrier device containing: a) transfectedprimary cells expressing exogenous DNA encoding erythropoietin, b)transfected secondary cells expressing exogenous DNA encodingerythropoietin, c) or both a) and b), wherein the barrier device is madeof a material which permits passage of erythropoietin into thecirculation or tissues of the mammal and prevents contact between theimmune system of the mammal and the transfected cells contained withinthe barrier device to a sufficient extent to prevent a deleteriousimmune response by the mammal.
 106. A method of providing erythropoietinin an effective amount to a mammal, comprising introducing into themammal a DNA construct comprising exogenous DNA encoding erythropoietinand regulatory sequences sufficient for expression of erythropoietin incells of the mammal, wherein the DNA construct is taken up by cells ofthe mammal and is expressed therein.
 107. The method of claim 106wherein the DNA construct is introduced into the mammal by directinjection into muscle.